Nucleic acid molecules encoding potassium channel interactors and uses therefor

ABSTRACT

The invention provides isolated nucleic acids molecules, designated PCIP nucleic acid molecules, which encode proteins that bind potassium channels and modulate potassium channel mediated activities. The invention also provides antisense nucleic acid molecules, recombinant expression vectors containing PCIP nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a PCIP gene has been introduced or disrupted. The invention still further provides isolated PCIP proteins, fusion proteins, antigenic peptides and anti-PCIP antibodies. Diagnostic methods utilizing compositions of the invention are also provided.

RELATED APPLICATIONS

This application claims priority to U.S. provisional Application No.60/110,033, filed on Nov. 25, 1998, U.S. provisional Application No.60/109,333, filed on Nov. 20, 1998, U.S. provisional Application No.60/110,277, filed on Nov. 30, 1998. This Application is a CIP of U.S.patent application Ser. No.: 09/350,614, filed on Jul. 9, 1999, which isa CIP of U.S. patent application Ser. No.: 09/350,874, filed on Jul. 9,1999, which is a CIP of U.S. patent application Ser. No. 09/298,731filed Apr. 23, 1999, incorporated herein in their entirety by thisreference.

BACKGROUND OF THE INVENTION

Mammalian cell membranes are important to the structural integrity andactivity of many cells and tissues. Of particular interest in membranephysiology is the study of transmembrane ion channels which act todirectly control a variety of pharmacological, physiological, andcellular processes. Numerous ion channels have been identified includingcalcium, sodium, and potassium channels, each of which have beeninvestigated to determine their roles in vertebrate and insect cells.

Because of their involvement in maintaining normal cellular homeostasis,much attention has been given to potassium channels. A number of thesepotassium channels open in response to changes in the cell membranepotential. Many voltage-gated potassium channels have been identifiedand characterized by their electrophysiological and pharmacologicalproperties. Potassium currents are more diverse than sodium or calciumcurrents and are further involved in determining the response of a cellto external stimuli.

The diversity of potassium channels and their important physiologicalrole highlights their potential as targets for developing therapeuticagents for various diseases. One of the best characterized classes ofpotassium channels are the voltage-gated potassium channels. Theprototypical member of this class is the protein encoded by the Shakergene in Drosophila melanogaster. Proteins of the Shal or Kv4 family area type of voltage-gated potassium channels that underlies many of thenative A type currents that have been recorded from different primarycells. Kv4 channels have a major role in the repolarization of cardiacaction potentials. In neurons, Kv4 channels and the A currents they maycomprise play an important role in modulation of firing rate, actionpotential initiation and in controlling dendritic responses to synapticinputs.

The fundamental function of a neuron is to receive, conduct, andtransmit signals. Despite the varied purpose of the signals carried bydifferent classes of neurons, the form of the signal is always the sameand consists of changes in the electrical potential across the plasmamembrane of the neuron. The plasma membrane of a neuron containsvoltage-gated cation channels, which are responsible for propagatingthis electrical potential (also referred to as an action potential ornerve impulse) across and along the plasma membrane.

The Kv family of channels includes, among others: (1) thedelayed-rectifier potassium channels, which repolarize the membraneafter each action potential to prepare the cell to fire again; and (2)the rapidly inactivating (A-type) potassium channels, which are activepredominantly at subthreshold voltages and and act to reduce the rate atwhich excitable cells reach firing threshold. In addition to beingcritical for action potential conduction, Kv channels also control theresponse to depolarizing, e.g., synaptic, inputs and play a role inneurotransmitter release. As a result of these activities, voltage-gatedpotassium channels are key regulators of neuronal excitability (HilleB., Ionic Channels of Excitable Membranes, Second Edition, Sunderland,Mass.: Sinauer, (1992)).

There is tremendous structural and functional diversity within the Kvpotassium channel superfamily. This diversity is generated both by theexistence of multiple genes and by alternative splicing of RNAtranscripts produced from the same gene. Nonetheless, the amino acidsequences of the known Kv potassium channels show high similarity. Allappear to be comprised of four, pore forming α-subunits and some areknown to have four cytoplasmic (β-subunit) polypeptides (Jan L. Y. etal. (1990) Trends Neurosci 13:415-419, and Pongs, O. et al. (1995) SemNeurosci. 7:137-146). The known Kv channel (α-subunits fall into foursub-families named for their homology to channels first isolated fromDrosophila: the Kv1, or Shaker-related subfamily; the Kv2, orShab-related subfamily; the Kv3, or Shaw-related subfamily; and the Kv4,or Shal-related subfamily.

Kv4.2 and Kv4.3 are examples of Kv channel (α-subunits of theShal-related subfamily. Kv4.3 has a unique neuroanatomical distributionin that its mRNA is highly expressed in brainstem monoaminergic andforebrain cholinergic neurons, where it is involved in the release ofthe neurotransmitters dopamine, norepinephrine, serotonin, andacetylcholine.

This channel is also highly expressed in cortical pyramidal cells and ininterneurons. (Serdio P. et al. (1996) J. Neurophys 75:2174-2179).Interestingly, the Kv4.3 polypeptide is highly expressed in neuronswhich express the corresponding mRNA. The Kv4.3 polypeptide is expressedin the somatodendritic membranes of these cells, where it is thought tocontribute to the rapidly inactivating K+ conductance. Kv4.2 mRNA iswidely expressed in brain, and the corresponding polypeptide alsoappears to be concentrated in somatodendritic membranes where it alsocontributes to the rapidly inactivating K+ conductance (Sheng et al.(1992) Neuron 9:271-84). These somatodendritic A-type Kv channels, likeKv4.2 and Kv4.3, are likely involved in processes which underlielearning and memory, such as integration of sub-threshold synapticresponses and the conductance of back-propagating action potentials(Hoffman D. A. et al. (1997) Nature 387:869-875).

Thus, proteins which interact with and modulate the activity ofpotassium channel proteins e.g., potassium channels having a Kv4.2 orKv4.3 subunit, provide novel molecular targets to modulate neuronalexcitability, e.g., action potential conduction, somatodendriticexcitability and neurotransmitter release, in cells expressing thesechannels. In addition, detection of genetic lesions in the gene encodingthese proteins could be used to diagnose and treat central nervoussystem disorders such as epilepsy, anxiety, depression, age-relatedmemory loss, migraine, obesity, Parkinsons disease or Alzheimer'sdisease.

Table of Contents A. Summary of the Invention  -3- B. Brief Descriptionof the Drawings  -9- C. Detailed Description of the Invention -13- I.Isolated Nucleic Acid Molecules -23- II. Isolated PCIP Proteins andAnti-PCIP Antibodies -38- III. Recombinant Expression Vectors and HostCells -48- IV. Pharmaceutical Compositions -56- V Uses and Methods ofthe Invention -61- A. Screening Assays -62- B. Detection Assays -69- 1.Chromosome Mapping -69- 2. Tissue Typing -71- 3. Use of Partial PCIPSequences in -72- Forensic Biology C. Predictive Medicine -73- 1.Diagnostic Assays -73- 2. Prognostic Assays -75- 3. Monitoring ofEffects During Clinical Trials -79- D. Methods of Treatment -80- 1.Prophylactic Methods -81- 2. Therapeutic Methods -81- 3.Pharmacogenomics -83- D. Examples -86-

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel nucleic acid molecules which encode gene products that interactwith potassium channel proteins or possess substantial homology to thegene products of the invention that interact with potassium channelproteins (paralogs). Potassium channel proteins are, for example,potassium channels having a Kv4.2 or Kv4.3 subunit. The nucleic acidmolecules of the invention and their gene products are referred toherein as “Potassium Channel Interacting Proteins”, “PCIP”, or “KChIP”nucleic acid and protein molecules. The PCIP proteins of the presentinvention interact with, e.g., bind to a potassium channel protein,modulate the activity of a potassium channel protein, and/or modulate apotassium channel mediated activity in a cell, e.g., a neuronal cell.The PCIP molecules of the present invention are useful as modulatingagents to regulate a variety of cellular processes, e.g., neuronal cellprocesses. Accordingly, in one aspect, this invention provides isolatednucleic acid molecules encoding PCIP proteins or biologically activeportions thereof, as well as nucleic acid fragments suitable as primersor hybridization probes for the detection of PCIP-encoding nucleicacids.

In one embodiment, a PCIP nucleic acid molecule of the invention is atleast 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or moreidentical to the nucleotide sequence (e.g., to the entire length of thenucleotide sequence) shown in SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:69, or SEQ ID NO:71, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98936, 98937, 98938,98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948,98949, 98950, 98951, 98991, 98993, or 98994, or a complement thereof.

In another preferred embodiment, the isolated nucleic acid moleculeincludes the nucleotide sequence shown SEQ ID NO:1, SEQ ID NO:3 SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:69, or SEQ ID NO:71, or a complement thereof. Inanother preferred embodiment, the nucleic acid molecule includes afragment of at least 300, 350, 400, 426, 471, or 583 nucleotides of thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:69, or SEQ ID NO:71, or a complement thereof.

In another embodiment, a PCIP nucleic acid molecule includes anucleotide sequence encoding a protein having an amino acid sequencesufficiently identical to the amino acid sequence of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:70, or SEQ ID NO:72, or an amino acid sequence encoded by the DNAinsert of the plasmid deposited with ATCC as Accession Number 98936,98937, 98938, 98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946,98947, 98948, 98949, 98950, 98951, 98991, 98993, or 98994. In apreferred embodiment, a PCIP nucleic acid molecule includes a nucleotidesequence encoding a protein having an amino acid sequence at least 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to theamino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ ID NO:72, or theamino acid sequence encoded by the DNA insert of the plasmid depositedwith ATCC as Accession Number 98936, 98937, 98938, 98939, 98940, 98941,98942, 98943, 98944, 98945, 98946, 98947, 98948, 98949, 98950, 98951,98991, 98993, or 98994.

In another preferred embodiment, an isolated nucleic acid moleculeencodes the amino acid sequence of 1v, 9q, p19, W28559, KChIP4a,KChIP4b, 33b07, 1p, and rat 7s protein. In yet another preferredembodiment, the nucleic acid molecule includes a nucleotide sequenceencoding a protein having the amino acid sequence of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:70, or SEQ ID NO:72, or the amino acid sequence encoded by the DNAinsert of the plasmid deposited with ATCC as Accession Number 98936,98937, 98938, 98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946,98947, 98948, 98949, 98950, 98951, 98991, 98993, or 98994. In yetanother preferred embodiment, the nucleic acid molecule is at least 426,471, or 583 nucleotides in length and encodes a protein having a PCIPactivity (as described herein).

Another embodiment of the invention features nucleic acid molecules,preferably PCIP nucleic acid molecules, which specifically detect PCIPnucleic acid molecules relative to nucleic acid molecules encodingnon-PCIP proteins. For example, in one embodiment, such a nucleic acidmolecule is at least 426, 400-450, 471, 450-500, 500-550, 583, 550-600,600-650, 650-700, 700-750, 750-800 or more nucleotides in length andhybridizes under stringent conditions to a nucleic acid moleculecomprising the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3 SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:69, or SEQ ID NO:71, the nucleotide sequence of the DNAinsert of the plasmid deposited with ATCC as Accession Number 98936,98937, 98938, 98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946,98947, 98948, 98949, 98950, 98951, 98991, 98993, or 98994, or acomplement thereof. In preferred embodiments, the nucleic acid moleculesare at least 15 (e.g., contiguous) nucleotides in length and hybridizeunder stringent conditions to nucleotides 93-126, 360-462, 732-825,1028-1054, or 1517-1534 of SEQ ID NO:7. In other preferred embodiments,the nucleic acid molecules comprise nucleotides 93-126, 360-462,732-825, 1028-1054, or 1517-1534 of SEQ ID NO:7.

In other preferred embodiments, the nucleic acid molecules are at least15 (e.g., contiguous) nucleotides in length and hybridize understringent conditions to nucleotides 1-14, 49-116, 137-311, 345-410,430-482, 503-518, 662-693, 1406-1421, 1441-1457, 1478-1494, or 1882-1959of SEQ ID NO:13. In other preferred embodiments, the nucleic acidmolecules comprise nucleotides 1-14, 49-116, 137-311, 345-410, 430-482,503-518, 662-693, 1406-1421, 1441-1457, 1478-1494, or 1882-1959 of SEQID NO:13.

In preferred embodiments, the nucleic acid molecules are at least 15(e.g., contiguous) nucleotides in length and hybridize under stringentconditions to nucleotides 932-1527, 1548-1765, 1786-1871, 1908-2091,2259-2265, or 2630-2654 of SEQ ID NO:35. In other preferred embodiments,the nucleic acid molecules comprise nucleotides 932-1527, 1548-1765,1786-1871, 1908-2091, 2259-2265, or 2630-2654 of SEQ ID NO:35.

In other preferred embodiments, the nucleic acid molecule encodes anaturally occurring allelic variant of a polypeptide comprising theamino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ ID NO:72 or anamino acid sequence encoded by the DNA insert of the plasmid depositedwith ATCC as Accession Number 98936, 98937, 98938, 98939, 98940, 98941,98942, 98943, 98944, 98945, 98946, 98947, 98948, 98949, 98950, 98951,98991, 98993, or 98994, wherein the nucleic acid molecule hybridizes toa nucleic acid molecule comprising SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:69, or SEQ ID NO:71 under stringent conditions.

Another embodiment of the invention provides an isolated nucleic acidmolecule which is antisense to a PCIP nucleic acid molecule, e.g., thecoding strand of a PCIP nucleic acid molecule.

Another aspect of the invention provides a vector comprising a PCIPnucleic acid molecule. In certain embodiments, the vector is arecombinant expression vector. In another embodiment, the inventionprovides a host cell containing a vector of the invention. The inventionalso provides a method for producing a protein, preferably a PCIPprotein, by culturing in a suitable medium, a host cell, e.g., amammalian host cell such as a non-human mammalian cell, of the inventioncontaining a recombinant expression vector, such that the protein isproduced.

Another aspect of this invention features isolated or recombinant PCIPproteins and polypeptides. In one embodiment, the isolated protein,preferably a PCIP protein, includes at least one calcium binding domain.In a preferred embodiment, the protein, preferably a PCIP protein,includes at least one calcium binding domain and has an amino acidsequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or more identical to the amino acid sequence of SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:70, or SEQ ID NO:72, or the amino acid sequence encoded by the DNAinsert of the plasmid deposited with ATCC as Accession Number 98936,98937, 98938, 98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946,98947, 98948, 98949, 98950, 98951, 98991, 98993, or 98994. In anotherpreferred embodiment, the protein, preferably a PCIP protein, includesat least one calcium binding domain and modulates a potassium channelmediated activity. In yet another preferred embodiment, the protein,preferably a PCIP protein, includes at least one calcium binding domainand is encoded by a nucleic acid molecule having a nucleotide sequencewhich hybridizes under stringent hybridization conditions to a nucleicacid molecule comprising the nucleotide sequence of SEQ ID NO:1, SEQ IDNO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56,SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71.

In another embodiment, the invention features fragments of the proteinshaving the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ ID NO:72,wherein the fragment comprises at least 15 amino acids (e.g., contiguousamino acids) of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ IDNO:72, or an amino acid sequence encoded by the DNA insert of theplasmid deposited with the ATCC as Accession Number 98936, 98937, 98938,98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948,98949, 98950, 98951, 98991, 98993, or 98994. In another embodiment, theprotein, preferably a PCIP protein, has the amino acid sequence of SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:70, or SEQ ID NO:72.

In another embodiment, the invention features an isolated protein,preferably a PCIP protein, which is encoded by a nucleic acid moleculehaving a nucleotide sequence at least about 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 95% or more identical to a nucleotide sequence ofSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71, or acomplement thereof.

The proteins of the present invention or biologically active portionsthereof, can be operatively linked to a non-PCIP polypeptide (e.g.,heterologous amino acid sequences) to form fusion proteins. Theinvention further features antibodies, such as monoclonal or polyclonalantibodies, that specifically bind proteins of the invention, preferablyPCIP proteins. In addition, the PCIP proteins or biologically activeportions thereof can be incorporated into pharmaceutical compositions,which optionally include pharmaceutically acceptable carriers.

In another aspect, the present invention provides a method for detectingthe presence of a PCIP nucleic acid molecule, protein or polypeptide ina biological sample by contacting the biological sample with an agentcapable of detecting a PCIP nucleic acid molecule, protein orpolypeptide such that the presence of a PCIP nucleic acid molecule,protein or polypeptide is detected in the biological sample.

In another aspect, the present invention provides a method for detectingthe presence of PCIP activity in a biological sample by contacting thebiological sample with an agent capable of detecting an indicator ofPCIP activity such that the presence of PCIP activity is detected in thebiological sample.

In another aspect, the invention provides a method for modulating PCIPactivity comprising contacting a cell capable of expressing PCIP with anagent that modulates PCIP activity such that PCIP activity in the cellis modulated. In one embodiment, the agent inhibits PCIP activity. Inanother embodiment, the agent stimulates PCIP activity. In oneembodiment, the agent is an antibody that specifically binds to a PCIPprotein. In another embodiment, the agent modulates expression of PCIPby modulating transcription of a PCIP gene or translation of a PCIPmRNA. In yet another embodiment, the agent is a nucleic acid moleculehaving a nucleotide sequence that is antisense. to the coding strand ofa PCIP mRNA or a PCIP gene.

In one embodiment, the methods of the present invention are used totreat a subject having a disorder characterized by aberrant PCIP proteinor nucleic acid expression or activity by administering an agent whichis a PCIP modulator to the subject. In one embodiment, the PCIPmodulator is a PCIP protein. In another embodiment the PCIP modulator isa PCIP nucleic acid molecule. In yet another embodiment, the PCIPmodulator is a peptide, peptidomimetic, or other small molecule. In apreferred embodiment, the disorder characterized by aberrant PCIPprotein or nucleic acid expression is a CNS disorder.

The present invention also provides a diagnostic assay for identifyingthe presence or absence of a genetic alteration characterized by atleast one of (i) aberrant modification or mutation of a gene encoding aPCIP protein; (ii) mis-regulation of the gene; and (iii) aberrantpost-translational modification of a PCIP protein, wherein a wild-typeform of the gene encodes a protein with a PCIP activity.

In another aspect the invention provides a method for identifying acompound that binds to or modulates the activity of a PCIP protein, byproviding an indicator composition comprising a PCIP protein having PCIPactivity, contacting the indicator composition with a test compound, anddetermining the effect of the test compound on PCIP activity in theindicator composition to identify a compound that modulates the activityof a PCIP protein.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the cDNA sequence and predicted amino acid sequence ofhuman 1v. The nucleotide sequence corresponds to nucleic acids 1 to 1463of SEQ ID NO:1. The amino acid sequence corresponds to amino acids 1 to216 of SEQ ID NO:2.

FIG. 2 depicts the cDNA sequence and predicted amino acid sequence ofrat 1v. The nucleotide sequence corresponds to nucleic acids 1 to 1856of SEQ ID NO:3. The amino acid sequence corresponds to amino acids 1 to245 of SEQ ID NO:4.

FIG. 3 depicts the cDNA sequence and predicted amino acid sequence ofmouse 1v. The nucleotide sequence corresponds to nucleic acids 1 to 1907of SEQ ID NO:5. The amino acid sequence corresponds to amino acids 1 to216 of SEQ ID NO:6.

FIG. 4 depicts the cDNA sequence and predicted amino acid sequence ofrat 1vl. The nucleotide sequence corresponds to nucleic acids 1 to 1534of SEQ ID NO:7. The amino acid sequence corresponds to amino acids 1 to227 of SEQ ID NO:8.

FIG. 5 depicts the cDNA sequence and predicted amino acid sequence ofmouse 1vl. The nucleotide sequence corresponds to nucleic acids 1 to1540 of SEQ ID NO:9. The amino acid sequence corresponds to amino acids1 to 227 of SEQ ID NO:10.

FIG. 6 depicts the cDNA sequence and predicted amino acid sequence ofrat 1vn. The nucleotide sequence corresponds to nucleic acids 1 to 955of SEQ ID NO:11. The amino acid sequence corresponds to amino acids 1 to203 of SEQ ID NO:12.

FIG. 7 depicts the cDNA sequence and predicted amino acid sequence ofhuman 9ql. The nucleotide sequence corresponds to nucleic acids 1 to2009 of SEQ ID NO:13. The amino acid sequence corresponds to amino acids1 to 270 of SEQ ID NO:14.

FIG. 8 depicts the cDNA sequence and predicted amino acid sequence ofrat 9ql. The nucleotide sequence corresponds to nucleic acids 1 to 1247of SEQ ID NO:15. The amino acid sequence corresponds to amino acids 1 to257 of SEQ ID NO:16.

FIG. 9 depicts the cDNA sequence and predicted amino acid sequence ofmouse 9ql. The nucleotide sequence corresponds to nucleic acids 1 to2343 of SEQ ID NO:17. The amino acid sequence corresponds to amino acids1 to 270 of SEQ ID NO:18.

FIG. 10 depicts the cDNA sequence and predicted amino acid sequence ofhuman 9qm. The nucleotide sequence corresponds to nucleic acids 1 to1955 of SEQ ID NO:19. The amino acid sequence corresponds to amino acids1 to 252 of SEQ ID NO:20.

FIG. 11 depicts the cDNA sequence and predicted amino acid sequence ofrat 9qm. The nucleotide sequence corresponds to nucleic acids 1 to 2300of SEQ ID NO:21. The amino acid sequence corresponds to amino acids 1 to252 of SEQ ID NO:22.

FIG. 12 depicts the cDNA sequence and predicted amino acid sequence ofhuman 9qs. The nucleotide sequence corresponds to nucleic acids 1 to1859 of SEQ ID NO:23. The amino acid sequence corresponds to amino acids1 to 220 of SEQ ID NO:24.

FIG. 13 depicts the cDNA sequence and predicted amino acid sequence ofmonkey 9qs. The nucleotide sequence corresponds to nucleic acids 1 to2191 of SEQ ID NO:25. The amino acid sequence corresponds to amino acids1 to 220 of SEQ ID NO:26.

FIG. 14 depicts the cDNA sequence and predicted amino acid sequence ofrat 9qc. The nucleotide sequence corresponds to nucleic acids 1 to 2057of SEQ ID NO:27. The amino acid sequence corresponds to amino acids 1 to252 of SEQ ID NO:28.

FIG. 15 depicts the cDNA sequence and predicted amino acid sequence ofrat 8t. The nucleotide sequence corresponds to nucleic acids 1 to 1904of SEQ ID NO:29. The amino acid sequence corresponds to amino acids 1 to225 of SEQ ID NO:30.

FIG. 16 depicts the cDNA sequence and predicted amino acid sequence ofhuman p19. The nucleotide sequence corresponds to nucleic acids 1 to 619of SEQ ID NO:31. The amino acid sequence corresponds to amino acids 1 to200 of SEQ ID NO:32.

FIG. 17 depicts the cDNA sequence and predicted amino acid sequence ofrat p19 The nucleotide sequence corresponds to nucleic acids 1 to 442 ofSEQ ID NO:33. The amino acid sequence corresponds to amino acids 1 to109 of SEQ ID NO:34.

FIG. 18 depicts the cDNA sequence and predicted amino acid sequence ofmouse p19. The nucleotide sequence corresponds to nucleic acids 1 to2644 of SEQ ID NO:35. The amino acid sequence corresponds to amino acids1 to 256 of SEQ. ID NO:36.

FIG. 19 depicts the cDNA sequence and predicted amino acid sequence ofhuman W28559. The nucleotide sequence corresponds to nucleic acids 1 to380 of SEQ ID NO:37. The amino acid sequence corresponds to amino acids1 to 126 of SEQ ID NO:38.

FIG. 20 depicts the cDNA sequence and predicted amino acid sequence ofhuman P193. The nucleotide sequence corresponds to nucleic acids 1 to2176 of SEQ ID NO:39. The amino acid sequence corresponds to amino acids1 to 41 of SEQ ID NO:40.

FIG. 21 depicts a schematic representation of the rat 1v, the rat 9qm,and the mouse P19 proteins, aligned to indicate the conserved domainsamong these proteins.

FIG. 22 depicts the genomic DNA sequence of human 9q. FIG. 22A depictsexon 1 and its flanking intron sequences (SEQ ID NO:46). FIG. 22Bdepicts exons 2-11 and the flanking intron sequences (SEQ ID NO:47).

FIG. 23 depicts the cDNA sequence and predicted amino acid sequence ofmonkey KChIP4a. The nucleotide sequence corresponds to nucleic acids 1to 2413 of SEQ ID NO:48. The amino acid sequence corresponds to aminoacids 1 to 233 of SEQ ID NO:49.

FIG. 24 depicts the cDNA sequence and predicted amino acid sequence ofmonkey KChIP4b. The nucleotide sequence corresponds to nucleic acids 1to 1591 of SEQ ID NO:50. The amino acid sequence corresponds to aminoacids 1 to 233 of SEQ ID NO:51.

FIG. 25 depicts an alignment of KChIP4a, KChIP4b, 9ql, 1v, p19, andrelated human paralog (hsncspara) W28559. Amino acids identical to theconsensus are shaded in black, conserved amino acids are shaded in gray.

FIG. 26 depicts the cDNA sequence and predicted amino acid sequence ofrat 33b07. The nucleotide sequence corresponds to nucleic acids 1 to2051 of SEQ ID NO:52. The amino acid sequence corresponds to amino acids1 to 407 of SEQ ID NO:53.

FIG. 27 depicts the cDNA sequence and predicted amino acid sequence ofhuman 33b07. The nucleotide sequence corresponds to nucleic acids 1 to4148 of SEQ ID NO:54. The amino acid sequence corresponds to amino acids1 to 414 of SEQ ID NO:55.

FIG. 28 depicts the cDNA sequence and predicted amino acid sequence ofrat 1p. The nucleotide sequence corresponds to nucleic acids 1 to 2643of SEQ ID NO:56. The amino acid sequence corresponds to amino acids 1 to267 of SEQ ID NO:57.

FIG. 29 depicts the cDNA sequence and predicted amino acid sequence ofrat 7s. The nucleotide sequence corresponds to nucleic acids 1 to 2929of SEQ ID NO:58. The amino acid sequence corresponds to amino acids 1 to270 of SEQ ID NO:59.

FIG. 30 depicts the cDNA sequence and predicted amino acid sequence ofrat 29x. The nucleotide sequence corresponds to nucleic acids 1 to 1489of SEQ ID NO:60. The amino acid sequence corresponds to amino acids 1 to351 of SEQ ID NO:61.

FIG. 31 depicts the cDNA sequence of rat 25r. The nucleotide sequencecorresponds to nucleic acids 1 to 1194 of SEQ ID NO:62.

FIG. 32 depicts the cDNA sequence and predicted amino acid sequence ofrat 5p. The nucleotide sequence corresponds to nucleic acids 1 to 600 ofSEQ ID NO:63. The amino acid sequence corresponds to amino acids 1 to 95of SEQ ID NO:64.

FIG. 33 depicts the cDNA sequence and predicted amino acid sequence ofrat 7q. The nucleotide sequence corresponds to nucleic acids 1 to 639 ofSEQ ID NO:65. The amino acid sequence corresponds to amino acids 1 to212 of SEQ ID NO:66.

FIG. 34 depicts the cDNA sequence and predicted amino acid sequence ofrat 19r. The nucleotide sequence corresponds to nucleic acids 1 to 816of SEQ ID NO:67. The amino acid sequence corresponds to amino acids 1 to271 of SEQ ID NO:68.

FIG. 35 depicts the cDNA sequence and predicted amino acid sequence ofmonkey KChIP4c. The nucleotide sequence corresponds to nucleic acids 1to 2263 of SEQ ID NO:69. The amino acid sequence corresponds to aminoacids 1 to 229 of SEQ ID NO:70.

FIG. 36 depicts the cDNA sequence and predicted amino acid sequence ofmonkey KChIP4d. The nucleotide sequence corresponds to nucleic acids 1to 2259 of SEQ ID NO:71. The amino acid sequence corresponds to aminoacids 1 to 250 of SEQ ID NO:72.

FIG. 37 depicts an alignment of KChIP4a, KChIP4b, KChIP4c, and KChIP4d.

FIG. 38 depicts a graph showing the current traces from CHO cells whichexpress Kv4.2 with or without KChIP2 (9ql). Cells are voltage clamped at−80 mV and stepped from −60 mV to +50 mV for 200 ms. Peak currentamplitudes at the various test voltages are shown in the right panel.FIG. 38 further depicts a table showing the amplitude and kineticeffects of KChIP2 (9ql) on Kv4.2. KchIP2 expression alters the peakcurrent amplitude, inactivation and recovery from inactivation timeconstants, and activation V_(1/2.)

FIG. 39 depicts a graph showing the current traces from CHO cells whichexpress Kv4.2 with or without KChIP3 (p19). Cells are voltage clamped at−80 mV and stepped from −60 mV to +50 mV for 200 ms. Peak currentamplitudes at the various test voltages are shown in the right panel.FIG. 39 further depicts a table showing the amplitude and kineticeffects of KchIP3 (p19) on Kv4.2. KchIP3 causes alterations in peakcurrent and inactivation and recovery from inactivation time constants.

FIG. 40 depicts results from electrophysiological experimentsdemonstrating that coexpression of KChIP1 dramatically alters thecurrent density and kinetics of Kv4.2 channels expressed in CHO cells.

FIG. 40A depicts current traces from a Kv4.2 transfected CHO cell.Current was evoked by depolarizing the cell sequentially from a holdingpotential of −80 mV to test potentials from −60 to 50 mV. Current tracesare leak subtracted using a p/5 protocol. The current axis is shown atthe same magnification as in (b) to emphasize the change in currentamplitudes. Inset- Single current trace at 50 mV at an expanded currentaxis to show the kinetics of current activation and inactivation.

FIG. 40B depicts current traces as in (a), but from a cell transfectedwith equal amounts of DNA for Kv4.2 and KChIP1.

FIG. 40C depicts peak current amplitude at all voltages from cellstransfected with Kv4.2 alone (n=11) or cotransfected with KChIP1 (n=9).

FIGS. 40D and 40E depict recovery from inactivation using a two pulseprotocol. Kv4.2 alone (D) or coexpressed with KChIP1 (E) is driven intothe inactivated state using a first pulse to 50 mV, then a second pulseto 50 mV is applied at varying times after the first pulse. Holdingpotential is −80 mV before and after all pulses.

FIG. 40F depicts a summary of the percentage the peak current recoversbetween pulses for Kv4.2 (n=8) and Kv4.2 plus KChIP1 (n=5) transfectedcells. The time constant of recovery from inactivation is fit to asingle exponential.

FIG. 41 depicts an alignment of human KChIP family members with closelyrelated members of the recoverin family of Ca 2+sensing proteins.(HIP:human hippocalcin; NCS1:rat neuronal calcium sensor 1). Thealignment was performed using the MegAlign program for Macintosh(version 4.00 from DNASTAR) using the Clustal method with the PAM250residue weight table and default parameters, and shaded using BOXSHADES.Residues identical to the consensus are shaded black, conservativesubstitutions are shaded grey. X, Y, Z and −X, −Y, −Z denote thepositions of residues which are responsible for binding to the calciumion in the EF hand.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based, at least in part, on the discovery ofnovel nucleic acid molecules which encode gene products that interactwith potassium channel proteins or possess substantial homology to thegene products of the invention that interact with potassium channelproteins (paralogs). Potassium channel proteins are, for example,potassium channels having a Kv4.2 or Kv4.3 subunit. The nucleic acidmolecules of the invention and their gene products are referred toherein as “Potassium Channel Interacting Proteins”, “PCIP”, or “KChIP”nucleic acid and protein molecules. Preferably, the PCIP proteins of thepresent invention interact with, e.g., bind to a potassium channelprotein, modulate the activity of a potassium channel protein, and/ormodulate a potassium channel mediated activity in a cell, e.g., aneuronal cell.

As used herein, the term “PCIP family” when referring to the protein andnucleic acid molecules of the invention is intended to mean two or moreproteins or nucleic acid molecules having a PCIP activity as definedherein. Such PCIP family members can be naturally or non-naturallyoccurring and can be from either the same or different species. Forexample, a PCIP family can contain a first protein of human origin, aswell as other, distinct proteins of human origin or alternatively, cancontain homologues of non-human origin.

As used interchangeably herein, a “PCIP activity”, “biological activityof PCIP” or “functional activity of PCIP”, refers to an activity exertedby a PCIP protein, polypeptide or nucleic acid molecule on a PCIPresponsive cell or on a PCIP protein substrate, as determined in vivo,or in vitro, according to standard techniques. In one embodiment, a PCIPactivity is a direct activity, such as an association with a PCIP-targetmolecule. As used herein, a “target molecule” or “binding partner” is amolecule with which a PCIP protein binds or interacts in nature, suchthat PCIP-mediated function is achieved. A PCIP target molecule can be anon-PCIP molecule or a PCIP protein or polypeptide of the presentinvention. In an exemplary embodiment, a PCIP target molecule is a PCIPligand. Alternatively, a PCIP activity is an indirect activity, such asa cellular signaling activity mediated by interaction of the PCIPprotein with a PCIP ligand. The biological activities of PCIP aredescribed herein.

For example, the PCIP proteins of the present invention can have one ormore of the following activities: (1) they can interact with (e.g., bindto) a potassium channel protein or portion thereof; (2) they canregulate the phosphorylation state of a potassium channel protein orportion thereof; (3) they can associate with (e.g., bind) calcium andcan, for example, act as calcium dependent kinases, e.g., phosphorylatea potassium channel or a G-protein coupled receptor in acalcium-dependent manner; (4) they can associate with (e.g., bind)calcium and can, for example, act in a calcium-dependent manner incellular processes, e.g., act as calcium dependent transcriptionfactors; (5) they can modulate a a potassium channel mediated activityin a cell (e.g., a neuronal cell such as a sensory neuron cell or amotor neuron cell) to, for example, beneficially affect the cell; (6)they can modulate chromatin formation in a cell, e.g., a neuronal cell;(7) they can modulate vesicular traffic and protein transport in a cell,e.g., a neuronal cell; (8) they can modulate cytokine signaling in acell, e.g., a neuronal cell; (9) they can regulate the association of apotassium channel protein or portion thereof with the cellularcytoskeleton; (10) they can modulate cellular proliferation; (11) theycan modulate the release of neurotransmitters; (12) they can modulatemembrane excitability; (13) they can influence the resting potential ofmembranes; (14) they can modulate wave forms and frequencies of actionpotentials; and (15) they can modulate thresholds of excitation.

As used herein, a “potassium channel” includes a protein or polypeptidethat is involved in receiving, conducting, and transmitting signals inan excitable cell. Potassium channels are typically expressed inelectrically excitable cells, e.g., neurons, cardiac, skeletal andsmooth muscle, renal, endocrine, and egg cells, and can formheteromultimeric structures, e.g., composed of pore-forming andcytoplasmic subunits. Examples of potassium channels include: (1) thevoltage-gated potassium channels, (2) the ligand-gated potassiumchannels, and (3) the mechanically-gated potassium channels. For adetailed description of potassium channels, see Kandel E. R. et al.,Principles of Neural Science, second edition, (Elsevier SciencePublishing Co., Inc., N.Y. (1985)), the contents of which areincorporated herein by reference. The PCIP proteins of the presentinvention have been shown to interact with, for example, potassiumchannels having a Kv4.3 subunit or a Kv4.2 subunit.

As used herein, a “potassium channel mediated activity” includes anactivity which involves a potassium channel, e.g., a potassium channelin a neuronal cell or a muscle cell, associated with receiving,conducting, and transmitting signals in, for example, the nervoussystem. Potassium channel mediated activities include release ofneurotransmitters, e.g., dopamine or norepinephrine, from cells, e.g.,neuronal cells; modulation of resting potential of membranes, wave formsand frequencies of action potentials, and thresholds of excitation; andmodulation of processes such as integration of sub-threshold synapticresponses and the conductance of back-propagating action potentials in,for example, neuronal cells or muscle cells. As the PCIP proteins of thepresent invention modulate potassium channel mediated activities, theymay be useful as novel diagnostic and therapeutic agents for potassiumchannel associated disorders and/or nervous system related disorders.

As used herein, a “potassium channel associated disorder” includes adisorder, disease or condition which is characterized by a misregulationof a potassium channel mediated activity. Potassium channel associateddisorders can detrimentally affect conveyance of sensory impulses fromthe periphery to the brain and/or conductance of motor impulses from thebrain to the periphery; integration of reflexes; interpretation ofsensory impulses; and emotional, intellectual (e.g., learning andmemory), or motor processes. As used herein, a “nervous system relateddisorder” includes a disorder, disease or condition which affects thenervous system. Examples of potassium channel associated disorders andnervous system related disorders include cognitive disorders, e.g.,memory and learning disorders, such as amnesia, apraxia, agnosia,amnestic dysnomia, amnestic spatial disorientation, Kluver-Bucysyndrome, Alzheimer's related memory loss (Eglen R. M. (1996) Pharmacol.and Toxicol. 78(2):59-68; Perry E. K. (1995) Brain and Cognition28(3):240-58) and learning disability; disorders affectingconsciousness, e.g., visual hallucinations, perceptual disturbances, ordelerium associated with Lewy body dementia; schitzo-effective disorders(Dean B. (1996) Mol. Psychiatry 1(1):54-8), schizophrenia with moodswings (Bymaster F. P. (1997) J. Clin. Psychiatry 58 (suppl.10):28-36;Yeomans J. S. (1995) Neuropharmacol. 12(1):3-16; Reimann D. (1994) J.Psychiatric Res. 28(3):195-210), depressive illness (primary orsecondary); affective disorders (Janowsky D. S. (1994) Am. J. Med.Genetics 54(4):335-44); sleep disorders (Kimura F. (1997) J.Neurophysiol. 77(2):709-16), e.g., REM sleep abnormalities in patientssuffering from, for example, depression (Riemann D. (1994) J.Psychosomatic Res. 38 Suppl. 1:15-25; Bourgin P. (1995) Neuroreport6(3): 532-6), paradoxical sleep abnormalities (Sakai K. (1997) Eur. J.Neuroscience 9(3):415-23), sleep-wakefulness, and body temperature orrespiratory depression abnormalities during sleep (Shuman S. L. (1995)Am. J. Physiol. 269(2 Pt 2):R308-17; Mallick B. N. (1997) Brain Res.750(1-2):311-7). Other examples of nervous system related disordersinclude disorders affecting pain generation mechanisms, e.g., painrelated to irritable bowel syndrome (Mitch C. H. (1997) J. Med. Chem.40(4):538-46; Shannon H. E. (1997) J. Pharmac. and Exp. Therapeutics281(2):884-94; Bouaziz H. (1995) Anesthesia and Analgesia 80(6):1140-4;or Guimaraes A. P. (1994) Brain Res. 647(2):220-30) or chest pain;movement disorders (Monassi C. R. (1997) Physiol. and Behav.62(1):53-9), e.g., Parkinson's disease related movement disorders (FinnM. (1997) Pharmacol. Biochem. & Behavior 57(1-2):243-9; Mayorga A. J.(1997) Pharmacol Biochem. & Behavior 56(2):273-9); eating disorders,e.g., insulin hypersecretion related obesity (Maccario M. (1997) J.Endocrinol. Invest. 20(1):8-12; Premawardhana L. D. (1994) Clin.Endocrinol. 40(5): 617-21); drinking disorders, e.g., diabeticpolydipsia (Murzi E. (1997) Brain Res. 752(1-2):184-8; Yang X. (1994)Pharmacol. Biochem. & Behavior 49(1):1-6) neurodegenerative disorders,e.g., Alzheimer's disease, dementias related to Alzheimer's disease(such as Pick's disease), Parkinson's and other Lewy diffuse bodydiseases, multiple sclerosis, amyotrophic lateral sclerosis, progressivesupranuclear palsy, epilepsy, epileptic syndromes, andJakob-Creutzfieldt disease; psychiatric disorders, e.g., depression,schizophrenic disorders, Korsakoff s psychosis, mania, anxietydisorders, bipolar affective disorders, or phobic disorders;neurological disorders, e.g., migraine; spinal cord injury; stroke; andhead trauma.

Some members of a PCIP family may also have common structuralcharacteristics, such as a common structural domain or motif or asufficient amino acid or nucleotide sequence homology as defined herein.Such PCIP family members can be naturally or non-naturally occurring andcan be from either the same or different species. For example, a PCIPfamily can contain a first protein of human origin, as well as other,distinct proteins of human origin or alternatively, can containhomologues of non-human origin.

For example, members of a PCIP family which have common structuralcharacteristics, may comprise at least one “calcium binding domain”. Asused herein, the term “calcium binding domain” includes an amino aciddomain, e.g., an EF hand (Baimbridge K. G. et al. (1992) TINS 15(8):303-308), which is involved in calcium binding. Preferably, a calciumbinding domain has a sequence, which is substantially identical to theconsensus sequence:

EO..OO..ODKDGDG.O...EF..OO.  (SEQ ID NO:41).

O can be I, L, V or M, and “.” indicates a position with no stronglypreferred residue. Each residue listed is present in more than 25% ofsequences, and those underlined are present in more than 80% ofsequences. Amino acid residues 126-154 and 174-202 of the human 1vprotein, amino acid residues 126-154 and 174-202 of the rat 1v protein,amino acid residues 137-165 and 185-213 of the rat 1vl protein, aminoacid residues 142-170 of the rat 1vn protein, amino acid residues126-154 and 174-202 of the mouse 1v protein, amino acid residues 137-165and 185-213 of the mouse 1vl protein, amino acid residues 144-172,180-208, and 228-256 of the human 9ql protein, amino acid residues126-154, 162-190, and 210-238 of the human 9qm protein, amino acidresidues 94-122, 130-158, and 178-206 of the human 9qs protein, aminoacid residues 126-154, 162-190, and 210-238 of the rat 9qm protein,amino acid residues 131-159, 167-195, and 215-243 of the rat 9qlprotein, amino acid residues 126-154, 162-190, and 210-238 of the rat9qc protein, amino acid residues 99-127, 135-163, and 183-211 of the rat8t protein, amino acid residues 144-172, 180-208, and 228-256 of themouse 9ql protein, amino acid residues 94-122, 130-158, and 178-206 ofthe monkey 9qs protein, amino acid residues 94-122, 130-158, and 178-206of the human p19 protein, amino acid residues 19-47 and 67-95 of the ratp19 protein, and amino acid residues 130-158, 166-194, and 214-242 ofthe mouse p19 protein comprise calcium binding domains (EF hands) (seeFIG. 21). Amino acid residues 116-127 and 152-163 of the monkey KChIP4aand KChIP4b proteins comprise calcium binding domains.

In another embodiment, the isolated PCIP proteins of the presentinvention are identified based on the presence of at least one conservedcarboxyl-terminal domain which includes an amino acid sequence of about100-200 amino acid residues in length, preferably 150-200 amino acidresidues in length, and more preferably 185 amino acid residues inlength, and which includes three EF hands. PCIP proteins of the presentinvention preferably contain a carboxyl-terminal domain which is atleast about 70%, 71%, 74%, 75%, 76%, 80%, or more identical to thecarboxyl terminal 185 amino acid residues of rat 1v, rat 9q, or mousep19 (see FIGS. 21, 25, and 41).

Members of the PCIP family which also have common structuralcharacteristics are listed in Table I and described below. The inventionprovides full length human, mouse, and rat 1v cDNA clones, full lengthmouse and rat cDNA clones of 1v splice variant 1vl, a partial rat cDNAclone of 1v splice variant 1vn, and the proteins encoded by these cDNAs.The invention further provides full length human and mouse and partialrat 9ql cDNA clones, full length human and rat cDNA clones of 9ql splicevariant 9qm, full length human and monkey cDNA clones of 9ql splicevariant 9qs, a full length rat cDNA clone of 9ql splice variant 9qc, apartial rat cDNA clone of 9ql splice variant 8t, and the proteinsencoded by these cDNAs. The invention also provides full length mouseand human and partial rat p19 cDNA clones and the proteins encoded bythese cDNAs. A full length human cDNA clone of p19 is provided, and apartial clone p193, representing the 3′ end of the human p19 cDNA. Inaddition, the invention provides a partial human W28559 cDNA clone andthe protein encoded by this cDNA. The invention further provides a fulllength monkey clone, KChIP4a, and a corresponding full length splicevariant, KChIP4b and the proteins encoded by these cDNAs.

Other members of the PCIP family, e.g., members of the PCIP family whichdo not have common structural characteristics, are listed in Table IIand are described below. The present invention provides a full lengthhuman and a partial length rat 33b07 clone and the proteins encoded bythese cDNAs. The present invention further provides partial length rat1p clone and the protein encoded by this cDNA. In addition, the presentinvention provides a partial length rat 7s clone and the protein encodedby this cDNA.

The present invention further provides PCIP family members whichrepresent previously identified cDNAs (29x, 25r, 5p, 7q, and 19r). Thesepreviously identified cDNAs are identified herein as PCIP familymembers, i.e., as molecules which have a PCIP activity, as describedherein. Accordingly, the present invention provides methods for usingthese previously identified cDNAs, e.g., methods for using these cDNAsin the screening assays, the diagnostic assays, the prognostic assays,and the methods of treatment described herein.

The PCIP molecules of the present invention were initially identifiedbased on their ability, as determined using yeast two-hybrid assays(described in detail in Example 1), to interact with the amino-terminal180 amino acids of rat Kv4.3 subunit. Further binding studies with otherpotassium subunits were performed to demonstrate specificity of the PCIPfor Kv4.3 and Kv4.2. In situ localization, immuno-histochemical methods,co-immunoprecipitation and patch clamping methods were then used toclearly demonstrate that the PCIPs of the present invention interactwith and modulate the activity of potassium channels, particularly thosecomprising a 4.3 or 4.2 subunit.

Several novel human, mouse, monkey, and rat PCIP family members havebeen identified, referred to herein as 1v, 9q, p19, W28559, KChIP4,33b07, 1p, and rat 7s proteins and nucleic acid molecules. The human,rat, and mouse cDNAs encoding the 1v polypeptide are represented by SEQID NOs:1, 3, and 5, and shown in FIGS. 1, 2, and 3, respectively. In thebrain, 1v mRNA is highly expressed in neocortical and hippocampalinterneurons, in the thalamic reticular nucleus and medial habenula, inbasal forebrain and striatal cholinergic neurons, in the superiorcolliculus, and in cerebellar granule cells. The 1v polypeptide ishighly expressed in the somata, dendrites, axons and axon terminals ofcells that express 1v mRNA. Splice variants of the 1v gene have beenidentified in rat and mouse and are represented by SEQ ID NOs: 7, 9, and11 and shown in FIGS. 4, 5, and 6, respectively. 1v polypeptideinteracts with potassium channels comprising Kv4.3 or kv4.2 subunits,but not with Kv1.1 subunits. As determined by Northern blot, the 1vtranscripts (mRNA) are expressed predominantly in the brain

The 8t cDNA (SEQ ID NO: 29) encodes a polypeptide having a molecularweight of approximately 26 kD corresponding to SEQ ID NO:30 (see FIG.15). The 8t polypeptide interacts with potassium channel comprisingKv4.3 or Kv4.2 subunits, but not with Kv1.1 subunits. As determined byNorthern blot and in situ data, the 8t mRNA is expressed predominantlyin the heart and the brain. The 8t cDNA is a splice variant of 9q.

Human, rat, monkey, and mouse 9q cDNA were also isolated. Splicevariants include human 9ql (SEQ ID NO:13; FIG. 7) rat 9ql (SEQ ID NO:15;FIG. 8), mouse 9ql (SEQ ID NO:17; FIG. 9), human 9qm (SEQ ID NO:19; FIG.10), rat 9qm (SEQ ID NO:21; FIG. 11), human 9qs (SEQ ID NO:23; FIG. 12),monkey 9qs (SEQ ID NO:25; FIG. 13), and rat 9qc (SEQ ID NO:27; FIG. 14).The genomic DNA sequence of 9q has also be determined. Exon 1 and itsflanking intron sequences (SEQ ID NO:46) are shown in FIG. 22A. Exons2-11 and the flanking intron sequences (SEQ ID NO:47) are shown in FIG.22B. 9q polypeptides interact with potassium channels comprising Kv4.3or Kv4.2 subunits, but not with Kv1.1 subunits. As determined byNorthern blot and in situ data, the 9q proteins are expressedpredominantly in the heart and the brain. In the brain, 9q mRNA ishighly expressed in the neostriatum, hippocampal formation, neocorticalpyramidal cells and interneurons, and in the thalamus, superiorcolliculus, and cerebellum.

Human, rat, and mouse P19 cDNA was also isolated. Human P19 is shown inSEQ ID NO:31 and FIG. 16; and in SEQ ID NO:39 and FIG. 20 (the 3′sequence). Rat P19 is shown in SEQ ID NO:33 and FIG. 17, and mouse P19is shown in SEQ ID NO:35 and FIG. 18. P19 polypeptides interact withpotassium channels comprising Kv4.3 or Kv4.2 subunits, but not withKv1.1 subunits. As determined by Northern blot analysis, the P19transcripts (mRNA) are expressed predominantly in the brain.

A partial human paralog of the PCIP molecules was also identified. Thisparalog is referred to herein as W28559 and is shown in SEQ ID NO:37 andFIG. 19.

Monkey KChIP4a and its splice variants KChIP4b, KChIP4c, and KChIP4dwere also identified. Monkey KChIP4a is shown in SEQ ID NO:48 and FIG.23. Monkey KChIP4b is shown in SEQ ID NO:50 and FIG. 24. Monkey KChIP4cis shown in SEQ ID NO:69 and FIG. 35. Monkey KChIP4d is shown in SEQ IDNO:71 and FIG. 36.

The nucleotide sequence of the full length rat 33b07 cDNA and thepredicted amino acid sequence of the rat 33b07 polypeptide are shown inFIG. 26 and in SEQ ID NOs:52 and 53, respectively. The rat 33b07 cDNAencodes a protein having a molecular weight of approximately 44.7 kD andwhich is 407 amino acid residues in length. Rat 33b07 binds rKv4.3N andrKv4.2N with slight preference for rKv4.2N in yeast 2-hybrid assays.

The nucleotide sequence of the fall length human 33b07 cDNA and thepredicted amino acid sequence of the human 33b07 polypeptide are shownin FIG. 27 and in SEQ ID NOs:54 and 55, respectively.

The nucleotide sequence of the partial length rat 1p cDNA and thepredicted amino acid sequence of the rat 1p polypeptide are shown inFIG. 28 and in SEQ ID NOs:56 and 57, respectively. The rat 1p cDNAencodes a protein having a molecular weight of approximately 28.6 kD andwhich is 267 amino acid residues in length. Rat 1p binds rKv4.3N andrKv4.2N with slight preference for rKv4.3N in yeast two-hybrid assays.

The nucleotide sequence of the partial length rat 7s cDNA and thepredicted amino acid sequence of the rat 7s polypeptide are shown inFIG. 29 and in SEQ ID NOs:58 and 59, respectively. The rat 7s cDNAencodes a protein having a molecular weight of approximately 28.6 kD andwhich is 270 amino acid residues in length. Rat 7s binds rKv4.3N andrKv4.2N with preference for rKv4.3N in yeast two-hybrid assays.

The sequences of the present invention are summarized below, in Tables Iand II.

TABLE I Novel Polynucleotides and Polypeptides of the Present Invention(full length except where noted) Nucleic SEQ SEQ Acid Mole- ID NO: IDNO: PCIP cule Form Source DNA PROTEIN ATCC 1v 1v human  1  2 98994 or (225-875)* KChIP1 1v rat  3  4 98946 (210-860) 1v mouse  5  6 98945 (477-1127) 1vl rat  7  8 98942  (31-714) 1vl mouse  9 10 98943 (77-760) 1vn rat 11 12 98944 (partial) (345-955) 9q Genomic human 46 orDNA se- KChIP2 quence (Exon 1 and flanking intron sequences) Genomichuman 47 DNA sequence (Exons 2-11 and flanking intron sequences) 9qlhuman 13 14 98993 (207-1019) 98991 9ql rat 15 16 98948 (partial) (2-775) 9ql mouse 17 18 98937 (181-993) 9qm human 19 20 98993 (207-965)98991 9qm rat 21 22 98941 (214-972) 9qs human 23 24 98951 (207-869) 9qsmonkey 25 26 98950 (133-795) 9qc rat 27 28 98947 (208-966) 8t rat 29 3098939 (partial)  (1-678) p19 p19 Human 31 32 PTA-316 or  (1-771) KChIP3p19 rat 33 34 98936 (partial)  (1-330) p19 mouse 35 36 98940  (49-819)p193 Human 39 40 98949 (partial)  (2-127) W28559 W28559 human 37 38(partial)  (1-339) KChIP4 KChIP4a Monkey 48 49 (265-966) KChIP4b Monkey50 51 C-terminal (265-966) splice variant KChIP4c Monkey 69 70 splicevariant (122-811) KChIP4d Monkey 71 72 splice variant  (64-816) *Thecoordinates of the coding sequence are shown in parenthesis. The firstcolumn indicates the PCIPs which were identified and column 2 indicatesthe various nucleic acid forms identified for each PCIP.

TABLE II Polynucleotides and Polypeptides of the Present Invention (fulllength except where noted) SEQ SEQ Nucleic Acid ID NO: ID NO: PCIPMolecule Form Source DNA PROTEIN ATCC 33b07 33b07 Human 52 53 PTA-316Novel (88-1332) 33b07 Rat 54 55 (85-1308) 1p 1p Rat 56 57 Novel(partial) (1-804) 7s 7s Rat 58 59 Novel (partial) (1-813) 29x 29x Rat 6061 (433-1071) 25r Rat 62 splice variant (130-768)  of 29x 5p 5p Rat 6364 (52-339) 7q 7q Rat 65 66  (1-639) 19r 19r Rat 67 68  (1-816) *Thecoordinates of the coding sequence are shown in parenthesis. The firstcolumn indicates the four families of PCIPs which were identified andcolumn 2 indicates the various nucleic acid forms identified for eachfamily. Novel molecules are also indicated.

Plasmids containing the nucleotide sequences encoding human, rat andmonkey PCIPs were deposited with American Type Culture Collection(ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, on Nov.17, 1998, and assigned the Accession Numbers described above. Thesedeposits will be maintained under the terms of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. These deposits were made merely as aconvenience for those of skill in the art and are not an admission thata deposit is required under 35 U.S.C. §112.

Clones containing cDNA molecules encoding human p19 (clone EphP19) andhuman 33b07 (clone Eph33b07) were deposited with American Type CultureCollection (Manassas, Va.) on Jul. 8, 1998 as Accession Number PTA-316,as part of a composite deposit representing a mixture of two strains,each carrying one recombinant plasmid harboring a particular cDNA clone.(The ATCC strain designation for the mixture of hP19 and h33b07 isEphP19h33b07mix).

To distinguish the strains and isolate a strain harboring a particularcDNA clone, an aliquot of the mixture can be streaked out to singlecolonies on LB plates supplemented with 100 ug/ml ampicillin, singlecolonies grown, and then plasmid DNA extracted using a standardminipreparation procedure. Next, a sample of the DNA minipreparation canbe digested with NotI and the resultant products resolved on a 0.8%agarose gel using standard DNA electrophoresis conditions. The digestgives the following band patterns: EphP19: 7 kb9 (single band),Eph33b07: 5.8 kb (single band).

Various aspects of the invention are described in further detail in thefollowing subsections:

I. Isolated Nucleic Acid Molecules

One aspect of the invention pertains to isolated nucleic acid moleculesthat encode PCIP proteins or biologically active portions thereof, aswell as nucleic acid fragments sufficient for use as hybridizationprobes to identify PCIP-encoding nucleic acid molecules (e.g., PCIPmRNA) and fragments for use as PCR primers for the amplification ormutation of PCIP nucleic acid molecules. As used herein, the term“nucleic acid molecule” is intended to include DNA molecules (e.g., cDNAor genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA orRNA generated using nucleotide analogs. The nucleic acid molecule can besingle-stranded or double-stranded, but preferably is double-strandedDNA.

An “isolated” nucleic acid molecule is one which is separated from othernucleic acid molecules which are present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. For example, invarious embodiments, the isolated PCIP nucleic acid molecule can containless than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb ofnucleotide sequences which naturally flank the nucleic acid molecule ingenomic DNA of the cell from which the nucleic acid is derived.Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule,can be substantially free of other cellular material, or culture mediumwhen produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g., a nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:69, or SEQ ID NO:71, or the nucleotide sequence of theDNA insert of the plasmid deposited with ATCC as Accession Number 98936,98937, 98938, 98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946,98947, 98948, 98949, 98950, 98951, 98991, 98993, or 98994, or a portionthereof, can be isolated using standard molecular biology techniques andthe sequence information provided herein. Using all or portion of thenucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:69, or SEQ ID NO:71, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98936, 98937, 98938,98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948,98949, 98950, 98951, 98991, 98993, or 98994, as a hybridization probe,PCIP nucleic acid molecules can be isolated using standard hybridizationand cloning techniques (e.g., as described in Sambrook, J., Fritsh, E.F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989).

Moreover, a nucleic acid molecule encompassing all or a portion of SEQID NO: 1, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98936, 98937, 98938, 98939, 98940, 98941, 98942,98943, 98944, 98945, 98946, 98947, 98948, 98949, 98950, 98951, 98991,98993, or 98994 can be isolated by the polymerase chain reaction (PCR)using synthetic oligonucleotide primers designed based upon the sequenceof SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98936, 98937, 98938, 98939, 98940, 98941, 98942,98943, 98944, 98945, 98946, 98947, 98948, 98949, 98950, 98951, 98991,98993, or 98994.

A nucleic acid of the invention can be amplified using cDNA, mRNA oralternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to PCIP nucleotide sequencescan be prepared by standard synthetic techniques, e.g., using anautomated DNA synthesizer.

In a preferred embodiment, an isolated nucleic acid molecule of theinvention comprises the nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56,SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71, or the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number98936, 98937, 98938, 98939, 98940, 98941, 98942, 98943, 98944, 98945,98946, 98947, 98948, 98949, 98950, 98951, 98991, 98993, or 98994, or aportion of any of these nucleotide sequences.

In another preferred embodiment, an isolated nucleic acid molecule ofthe invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SESQID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56 , SEQ ID NO:58, SEQ IDNO:69, or SEQ ID NO:71, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98936, 98937, 98938,98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948,98949, 98950, 98951, 98991, 98993, or 98994, or a portion of any ofthese nucleotide sequences. A nucleic acid molecule which iscomplementary to the nucleotide sequence shown in SEQ ID NO:1, SEQ IDNO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56,SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71, or the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number98936, 98937, 98938, 98939, 98940, 98941, 98942, 98943, 98944, 98945,98946, 98947, 98948, 98949, 98950, 98951, 98991, 98993, or 98994, is onewhich is sufficiently complementary to the nucleotide sequence shown inSEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ IDNO:9, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98936, 98937, 98938, 98939,98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948, 98949,98950, 98951, 98991, 98993, or 98994, such that it can hybridize to thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:69, or SEQ ID NO:71, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98936, 98937, 98938,98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948,98949, 98950, 98951, 98991, 98993, or 98994, thereby forming a stableduplex.

In still another preferred embodiment, an isolated nucleic acid moleculeof the present invention comprises a nucleotide sequence which is atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or moreidentical to the entire length of the nucleotide sequence shown in SEQID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71, or theentire length of the nucleotide sequence of the DNA insert of theplasmid deposited with ATCC as Accession Number 98936, 98937, 98938,98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948,98949, 98950, 98951, 98991, 98993, or 98994, or a portion of any ofthese nucleotide sequences.

Moreover, the nucleic acid molecule of the invention can comprise only aportion of the nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3 SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:69, or SEQ ID NO:71, or the nucleotide sequence of theDNA insert of the plasmid deposited with ATCC as Accession Number 98936,98937, 98938, 98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946,98947, 98948, 98949, 98950, 98951, 98991, 98993, or 98994, for example afragment which can be used as a probe or primer or a fragment encoding abiologically active portion of a PCIP protein. The nucleotide sequencedetermined from the cloning of the PCIP gene allows for the generationof probes and primers designed for use in identifying and/or cloningother PCIP family members, as well as PCIP homologues from otherspecies.

The probe/primer typically comprises substantially purifiedoligonucleotide. The oligonucleotide typically comprises a region ofnucleotide sequence that hybridizes under stringent conditions to atleast about 12 or 15, preferably about 20 or 25, more preferably about30, 35, 40, 45, 50, 55, 60, 65, or 75 consecutive nucleotides of a sensesequence of SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ IDNO:71, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98936, 98937, 98938, 98939,98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948, 98949,98950, 98951, 98991, 98993, or 98994, of an anti-sense sequence of SEQID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98936, 98937, 98938, 98939, 98940, 98941, 98942,98943, 98944, 98945, 98946, 98947, 98948, 98949, 98950, 98951, 98991,98993, or 98994, or of a naturally occurring allelic variant or mutantof SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98936, 98937, 98938, 98939, 98940, 98941, 98942,98943, 98944, 98945, 98946, 98947, 98948, 98949, 98950, 98951, 98991,98993, or 98994. In an exemplary embodiment, a nucleic acid molecule ofthe present invention comprises a nucleotide sequence which is 350-400,400-450, 450-500, 500-550, 550-600, 600-650, 650-700, 700-750, 750-800,800-850, 850-900, 949, 950-1000, or more nucleotides in length andhybridizes under stringent hybridization conditions to a nucleic acidmolecule of SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ IDNO:71, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98936, 98937, 98938, 98939,98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948, 98949,98950, 98951, 98991, 98993, or 98994.

Probes based on the PCIP nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, e.g., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which misexpress a PCIP protein, such as by measuring a level ofa PCIP-encoding nucleic acid in a sample of cells from a subject e.g.,detecting PCIP mRNA levels or determining whether a genomic PCIP genehas been mutated or deleted.

A nucleic acid fragment encoding a “biologically active portion of aPCIP protein” can be prepared by isolating a portion of the nucleotidesequence of SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ IDNO:71, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98936, 98937, 98938, 98939,98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948, 98949,98950, 98951, 98991, 98993, or 98994, which encodes a polypeptide havinga PCIP biological activity (the biological activities of the PCIPproteins are described herein), expressing the encoded portion of thePCIP protein (e.g., by recombinant expression in vitro) and assessingthe activity of the encoded portion of the PCIP protein.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3 SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:69, or SEQ ID NO:71 or the nucleotide sequence of theDNA insert of the plasmid deposited with 98936, 98937, 98938, 98939,98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948, 98949,98950, 98951, 98991, 98993, or 98994, due to degeneracy of the geneticcode and thus encode the same PCIP proteins as those encoded by thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:69, or SEQ ID NO:71 or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98936, 98937, 98938,98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948,98949, 98950, 98951, 98991, 98993, or 98994. In another embodiment, anisolated nucleic acid molecule of the invention has a nucleotidesequence encoding a protein having an amino acid sequence shown in SEQID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:70, or SEQ ID NO:72.

In addition to the PCIP nucleotide sequences shown in SEQ ID NO:1, SEQID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71, or the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98936, 98937, 98938, 98939, 98940, 98941, 98942, 98943,98944, 98945, 98946, 98947, 98948, 98949, 98950, 98951, 98991, 98993, or98994, it will be appreciated by those skilled in the art that DNAsequence polymorphisms that lead to changes in the amino acid sequencesof the PCIP proteins may exist within a population (e.g., the humanpopulation). Such genetic polymorphism in the PCIP genes may exist amongindividuals within a population due to natural allelic variation. Asused herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding a PCIPprotein, preferably a mammalian PCIP protein, and can further includenon-coding regulatory sequences, and introns.

Allelic variants of human PCIP include both functional andnon-functional PCIP proteins. Functional allelic variants are naturallyoccurring amino acid sequence variants of the human PCIP protein thatmaintain the ability to bind a PCIP ligand and/or modulate any of thePCIP activities described herein. Functional allelic variants willtypically contain only conservative substitution of one or more aminoacids of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ ID NO:72 or substitution,deletion or insertion of non-critical residues in non-critical regionsof the protein.

Non-functional allelic variants are naturally occurring amino acidsequence variants of the human PCIP protein that do not have the abilityto either bind a PCIP ligand and/or modulate any of the PCIP activitiesdescribed herein. Non-functional allelic variants will typically containa non-conservative substitution, a deletion, or insertion or prematuretruncation of the amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ IDNO:72 or a substitution, insertion or deletion in critical residues orcritical regions.

The present invention further provides non-human orthologues of thehuman PCIP protein. Orthologues of the human PCIP protein are proteinsthat are isolated from non-human organisms and possess the same PCIPligand binding and/or modulation of potassium channel mediatedactivities of the human PCIP protein. Orthologues of the human PCIPprotein can readily be identified as comprising an amino acid sequencethat is substantially identical to SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ IDNO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ IDNO:72.

Moreover, nucleic acid molecules encoding other PCIP family members and,thus, which have a nucleotide sequence which differs from the PCIPsequences of SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ IDNO:71, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98936, 98937, 98938, 98939,98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948, 98949,98950, 98951, 98991, 98993, or 98994 are intended to be within the scopeof the invention. For example, another PCIP cDNA can be identified basedon the nucleotide sequence of human PCIP. Moreover, nucleic acidmolecules encoding PCIP proteins from different species, and thus whichhave a nucleotide sequence which differs from the PCIP sequences of SEQID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71 or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98936, 98937, 98938, 98939, 98940, 98941, 98942,98943, 98944, 98945, 98946, 98947, 98948, 98949, 98950, 98951, 98991,98993, or 98994 are intended to be within the scope of the invention.For example, a mouse PCIP cDNA can be identified based on the nucleotidesequence of a human PCIP.

Nucleic acid molecules corresponding to natural allelic variants andhomologues of the PCIP cDNAs of the invention can be isolated based ontheir homology to the PCIP nucleic acids disclosed herein using thecDNAs disclosed herein, or a portion thereof, as a hybridization probeaccording to standard hybridization techniques under stringenthybridization conditions.

Accordingly, in another embodiment, an isolated nucleic acid molecule ofthe invention is at least 15, 20, 25, 30 or more nucleotides in lengthand hybridizes under stringent conditions to the nucleic acid moleculecomprising the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ IDNO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ IDNO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ IDNO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ IDNO:58, SEQ ID NO:69, or SEQ ID NO:71 or the nucleotide sequence of theDNA insert of the plasmid deposited with ATCC as Accession Number 98936,98937, 98938, 98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946,98947, 98948, 98949, 98950, 98951, 98991, 98993, or 98994. In otherembodiment, the nucleic acid is at least 30, 50, 100, 150, 200, 250,300, 307, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,949, or 950 nucleotides in length. As used herein, the term “hybridizesunder stringent conditions” is intended to describe conditions forhybridization and washing under which nucleotide sequences at least 60%identical to each other typically remain hybridized to each other.Preferably, the conditions are such that sequences at least about 70%,more preferably at least about 80%, even more preferably at least about85% or 90% identical to each other typically remain hybridized to eachother. Such stringent conditions are known to those skilled in the artand can be found in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6. A preferred, non-limiting example ofstringent hybridization conditions are hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by one or morewashes in 0.2× SSC, 0.1% SDS at 50° C., preferably at 55° C., and morepreferably at 60° C. or 65° C. Preferably, an isolated nucleic acidmolecule of the invention that hybridizes under stringent conditions tothe sequence of SEQ ID NO:1 corresponds to a naturally-occurring nucleicacid molecule. As used herein, a “naturally-occurring” nucleic acidmolecule refers to an RNA or DNA molecule having a nucleotide sequencethat occurs in nature (e.g., encodes a natural protein).

In addition to naturally-occurring allelic variants of the PCIPsequences that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequences of SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:69, or SEQ ID NO:71, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98936, 98937, 98938,98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948,98949, 98950, 98951, 98991, 98993, or 98994, thereby leading to changesin the amino acid sequence of the encoded PCIP proteins, withoutaltering the functional ability of the PCIP proteins. For example,nucleotide substitutions leading to amino acid substitutions at“non-essential” amino acid residues can be made in the sequence of SEQID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ IDNO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71, or thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98936, 98937, 98938, 98939, 98940, 98941, 98942,98943, 98944, 98945, 98946, 98947, 98948, 98949, 98950, 98951, 98991,98993, or 98994. A “non-essential” amino acid residue is a residue thatcan be altered from the wild-type sequence of PCIP (e.g., the sequenceof SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ IDNO:59, SEQ ID NO:70, or SEQ ID NO:72) without altering the biologicalactivity, whereas an “essential” amino acid residue is required forbiological activity. For example, amino acid residues that are conservedamong the PCIP proteins of the present invention, are predicted to beparticularly unamenable to alteration. Furthermore, additional aminoacid residues that are conserved between the PCIP proteins of thepresent invention and other members of the PCIP family of proteins arenot likely to be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding PCIP proteins that contain changes in amino acidresidues that are not essential for activity. Such PCIP proteins differin amino acid sequence from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ ID NO:72, yetretain biological activity. In one embodiment, the isolated nucleic acidmolecule comprises a nucleotide sequence encoding a protein, wherein theprotein comprises an amino acid sequence at least about 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or more identical to SEQ ID NO:2, SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:70, or SEQ ID NO:72.

An isolated nucleic acid molecule encoding a PCIP protein homologous tothe protein of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ ID NO:72 can be created byintroducing one or more nucleotide substitutions, additions or deletionsinto the nucleotide sequence of SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:69, or SEQ ID NO:71, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98936, 98937, 98938,98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948,98949, 98950, 98951, 98991, 98993, or 98994, such that one or more aminoacid substitutions, additions or deletions are introduced into theencoded protein. Mutations can be introduced into SEQ ID NO:1, SEQ IDNO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13,SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23,SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33,SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47,SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56,SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71, or the nucleotide sequenceof the DNA insert of the plasmid deposited with ATCC as Accession Number98936, 98937, 98938, 98939, 98940, 98941, 98942, 98943, 98944, 98945,98946, 98947, 98948, 98949, 98950, 98951, 98991, 98993, or 98994 bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a PCIP protein ispreferably replaced with another amino acid residue from the same sidechain family. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of a PCIP coding sequence, such asby saturation mutagenesis, and the resultant mutants can be screened forPCIP biological activity to identify mutants that retain activity.Following mutagenesis of SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:69, or SEQ ID NO:71, or the nucleotide sequence of the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98936, 98937, 98938,98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948,98949, 98950, 98951, 98991, 98993, or 98994, the encoded protein can beexpressed recombinantly and the activity of the protein can bedetermined.

In a preferred embodiment, a mutant PCIP protein can be assayed for theability to (1) interact with (e.g., bind to) a potassium channel proteinor portion thereof; (2) regulate the phosphorylation state of apotassium channel protein or portion thereof; (3) associate with (e.g.,bind) calcium and, for example, act as a calcium dependent kinase, e.g.,phosphorylate a potassium channel in a calcium-dependent manner; (4)associate with (e.g., bind) calcium and, for example, act as a calciumdependent transcription factor; (5) modulate a potassium channelmediated activity in a cell (e.g., a neuronal cell) to, for example,beneficially affect the cell; (6) modulate the release ofneurotransmitters; (7) modulate membrane excitability; (8) influence theresting potential of membranes; (9) modulate wave forms and frequenciesof action potentials; and (10) modulate thresholds of excitation.

In addition to the nucleic acid molecules encoding PCIP proteinsdescribed above, another aspect of the invention pertains to isolatednucleic acid molecules which are antisense thereto. An “antisense”nucleic acid comprises a nucleotide sequence which is complementary to a“sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bondto a sense nucleic acid. The antisense nucleic acid can be complementaryto an entire PCIP coding strand, or to only a portion thereof. In oneembodiment, an antisense nucleic acid molecule is antisense to a “codingregion” of the coding strand of a nucleotide sequence encoding PCIP. Theterm “coding region” refers to the region of the nucleotide sequencecomprising codons which are translated into amino acid residues. Inanother embodiment, the antisense nucleic acid molecule is antisense toa “noncoding region” of the coding strand of a nucleotide sequenceencoding PCIP. The term “noncoding region” refers to 5′ and 3′ sequenceswhich flank the coding region that are not translated into amino acids(i.e., also referred to as 5′ and 3′ untranslated regions).

Given the coding strand sequences encoding PCIP disclosed herein,antisense nucleic acids of the invention can be designed according tothe rules of Watson and Crick base pairing. The antisense nucleic acidmolecule can be complementary to the entire coding region of PCIP mRNA,but more preferably is an oligonucleotide which is antisense to only aportion of the coding or noncoding region of PCIP mRNA. For example, theantisense oligonucleotide can be complementary to the region surroundingthe translation start site of PCIP mRNA. An antisense oligonucleotidecan be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50nucleotides in length. An antisense nucleic acid of the invention can beconstructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art. For example, an antisense nucleicacid (e.g., an antisense oligonucleotide) can be chemically synthesizedusing naturally occurring nucleotides or variously modified nucleotidesdesigned to increase the biological stability of the molecules or toincrease the physical stability of the duplex formed between theantisense and sense nucleic acids, e.g., phosphorothioate derivativesand acridine substituted nucleotides can be used. Examples of modifiednucleotides which can be used to generate the antisense nucleic acidinclude 5-fluorouracil, 5-bromouracil, 5-chlorouracil 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject or generated in situ such that they hybridizewith or bind to cellular mRNA and/or genomic DNA encoding a PCIP proteinto thereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. The hybridization can be byconventional nucleotide complementarity to form a stable duplex, or, forexample, in the case of an antisense nucleic acid molecule which bindsto DNA duplexes, through specific interactions in the major groove ofthe double helix. An example of a route of administration of antisensenucleic acid molecules of the invention include direct injection at atissue site. Alternatively, antisense nucleic acid molecules can bemodified to target selected cells and then administered systemically.For example, for systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. Ribozymes are catalytic RNA molecules with ribonucleaseactivity which are capable of cleaving a single-stranded nucleic acid,such as an mRNA, to which they have a complementary region. Thus,ribozymes (e.g., hammerhead ribozymes (described in Haselhoff andGerlach (1988) Nature 334:585-591)) can be used to catalytically cleavePCIP mRNA transcripts to thereby inhibit translation of PCIP mRNA. Aribozyme having specificity for a PCIP-encoding nucleic acid can bedesigned based upon the nucleotide sequence of a PCIP cDNA disclosedherein (i.e., SEQ ID NO:1, SEQ ID NO:3 SEQ ID, NO:5, SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ IDNO:71, or the nucleotide sequence of the DNA insert of the plasmiddeposited with ATCC as Accession Number 98936, 98937, 98938, 98939,98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948, 98949,98950, 98951, 98991, 98993, or 98994). For example, a derivative of aTetrahymena L-19 IVS RNA can be constructed in which the nucleotidesequence of the active site is complementary to the nucleotide sequenceto be cleaved in a PCIP-encoding mRNA. See, e.g., Cech et al. U.S. Pat.No. 4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively,PCIP mRNA can be used to select a catalytic RNA having a specificribonuclease activity from a pool of RNA molecules. See, e.g., Bartel,D. and Szostak, J. W. (1993) Science 261:1411-1418.

Alternatively, PCIP gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the PCIP(e.g., the PCIP promoter and/or enhancers) to form triple helicalstructures that prevent transcription of the PCIP gene in target cells.See generally, Helene, C. (1991) Anticancer Drug Des. 6(6):569-84;Helene, C. et al. (1992) Ann. N. Y. Acad. Sci. 660:27-36; and Maher, L.J. (1992) Bioassays 14(12):807-15.

In yet another embodiment, the PCIP nucleic acid molecules of thepresent invention can be modified at the base moiety, sugar moiety orphosphate backbone to improve, e.g., the stability, hybridization, orsolubility of the molecule. For example, the deoxyribose phosphatebackbone of the nucleic acid molecules can be modified to generatepeptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & MedicinalChemistry 4 (1): 5-23). As used herein, the terms “peptide nucleicacids” or “PNAs” refer to nucleic acid mimics, e.g., DNA mimics, inwhich the deoxyribose phosphate backbone is replaced by a pseudopeptidebackbone and only the four natural nucleobases are retained. The neutralbackbone of PNAs has been shown to allow for specific hybridization toDNA and RNA under conditions of low ionic strength. The synthesis of PNAoligomers can be performed using standard solid phase peptide synthesisprotocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe etal. Proc. Natl. Acad. Sci. 93: 14670-675.

PNAs of PCIP nucleic acid molecules can be used in therapeutic anddiagnostic applications. For example, PNAs can be used as antisense orantigene agents for sequence-specific modulation of gene expression by,for example, inducing transcription or translation arrest or inhibitingreplication. PNAs of PCIP nucleic acid molecules can also be used in theanalysis of single base pair mutations in a gene, (e.g., by PNA-directedPCR clamping); as ‘artificial restriction enzymes’ when used incombination with other enzymes, (e.g., S1 nucleases (Hyrup B. (1996)supra)); or as probes or primers for DNA sequencing or hybridization(Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).

In another embodiment, PNAs of PCIP can be modified, (e.g., to enhancetheir stability or cellular uptake), by attaching lipophilic or otherhelper groups to PNA, by the formation of PNA-DNA chimeras, or by theuse of liposomes or other techniques of drug delivery known in the art.For example, PNA-DNA chimeras of PCIP nucleic acid molecules can begenerated which may combine the advantageous properties of PNA and DNA.Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNApolymerases), to interact with the DNA portion while the PNA portionwould provide high binding affinity and specificity. PNA-DNA chimerascan be linked using linkers of appropriate lengths selected in terms ofbase stacking, number of bonds between the nucleobases, and orientation(Hyrup B. (1996) supra). The synthesis of PNA-DNA chimeras can beperformed as described in Hyrup B. (1996) supra and Finn P. J. et al.(1996) Nucleic Acids Res. 24 (17): 3357-63. For example, a DNA chain canbe synthesized on a solid support using standard phosphoramiditecoupling chemistry and modified nucleoside analogs, e.g.,5′-(4-methoxytrityl)amino-5′-deoxy-thymidine phosphoramidite, can beused as a between the PNA and the 5′ end of DNA (Mag, M. et al. (1989)Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in astepwise manner to produce a chimeric molecule with a 5′ PNA segment anda 3′ DNA segment (Finn P. J. et al. (1996) supra). Alternatively,chimeric molecules can be synthesized with a 5′ DNA segment and a 3′ PNAsegment (Peterser, K. H. et al. (1975) Bioorganic Med. Chem. Lett. 5:1119-11124).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. US. 86:6553-6556;Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. W088/09810) or the blood-brain barrier (see, e.g., PCTPublication No. W089/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (See, e.g., Krolet al. (1988) Bio-Techniques 6:958-976) or intercalating agents. (See,e.g., Zon (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

II. Isolated PCIP Proteins and Anti-PCIP Antibodies

One aspect of the invention pertains to isolated PCIP proteins, andbiologically active portions thereof, as well as polypeptide fragmentssuitable for use as immunogens to raise anti-PCIP antibodies. In oneembodiment, native PCIP proteins can be isolated from cells or tissuesources by an appropriate purification scheme using standard proteinpurification techniques. In another embodiment, PCIP proteins areproduced by recombinant DNA techniques. Alternative to recombinantexpression, a PCIP protein or polypeptide can be synthesized chemicallyusing standard peptide synthesis techniques.

An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material or othercontaminating proteins from the cell or tissue source from which thePCIP protein is derived, or substantially free from chemical precursorsor other chemicals when chemically synthesized. The language“substantially free of cellular material” includes preparations of PCIPprotein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced. In oneembodiment, the language “substantially free of cellular material”includes preparations of PCIP protein having less than about 30% (by dryweight) of non-PCIP protein (also referred to herein as a “contaminatingprotein”), more preferably less than about 20% of non-PCIP protein,still more preferably less than about 10% of non-PCIP protein, and mostpreferably less than about 5% non-PCIP protein. When the PCIP protein orbiologically active portion thereof is recombinantly produced, it isalso preferably substantially free of culture medium, i.e., culturemedium represents less than about 20%, more preferably less than about10%, and most preferably less than about 5% of the volume of the proteinpreparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of PCIP protein in which the protein isseparated from chemical precursors or other chemicals which are involvedin the synthesis of the protein. In one embodiment, the language“substantially free of chemical precursors or other chemicals” includespreparations of PCIP protein having less than about 30% (by dry weight)of chemical precursors or non-PCIP chemicals, more preferably less thanabout 20% chemical precursors or non-PCIP chemicals, still morepreferably less than about 10% chemical precursors or non-PCIPchemicals, and most preferably less than about 5% chemical precursors ornon-PCIP chemicals.

As used herein, a “biologically active portion” of a PCIP proteinincludes a fragment of a PCIP protein which participates in aninteraction between a PCIP molecule and a non-PCIP molecule.Biologically active portions of a PCIP protein include peptidescomprising amino acid sequences sufficiently identical to or derivedfrom the amino acid sequence of the PCIP protein, e.g., the amino acidsequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38;SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55,SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ ID NO:72, which includeless amino acids than the full length PCIP proteins, and exhibit atleast one activity of a PCIP protein. Typically, biologically activeportions comprise a domain or motif with at least one activity of thePCIP protein, e.g., binding of a potassium channel subunit. Abiologically active portion of a PCIP protein can be a polypeptide whichis, for example, 10, 25, 50, 100, 200, or more amino acids in length.Biologically active portions of a PCIP protein can be used as targetsfor developing agents which modulate a potassium channel mediatedactivity.

In one embodiment, a biologically active portion of a PCIP proteincomprises at least one calcium binding domain.

It is to be understood that a preferred biologically active portion of aPCIP protein of the present invention may contain at least one of theabove-identified structural domains. A more preferred biologicallyactive portion of a PCIP protein may contain at least two of theabove-identified structural domains. Moreover, other biologically activeportions, in which other regions of the protein are deleted, can beprepared by recombinant techniques and evaluated for one or more of thefunctional activities of a native PCIP protein.

In a preferred embodiment, the PCIP protein has an amino acid sequenceshown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ ID NO:72. In otherembodiments, the PCIP protein is substantially homologous to SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49,SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59,SEQ ID NO:70, or SEQ ID NO:72, and retains the functional activity ofthe protein of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ ID NO:72, yet differs in aminoacid sequence due to natural allelic variation or mutagenesis, asdescribed in detail in subsection I above. Accordingly, in anotherembodiment, the PCIP protein is a protein which comprises an amino acidsequence at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%or more identical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28,SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38,SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55,SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ ID NO:72.

Isolated proteins of the present invention, preferably 1v, 9q, p19,W28559, KChIP4a, KChIP4b, 33b07, 1p, or 7s proteins, have an amino acidsequence sufficiently identical to the amino acid sequence of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:49,SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59,SEQ ID NO:70, or SEQ ID NO:72 or are encoded by a nucleotide sequencesufficiently identical to SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:69, or SEQ ID NO:71. As used herein, the term “sufficientlyidentical” refers to a first amino acid or nucleotide sequence whichcontains a sufficient or minimum number of identical or equivalent(e.g., an amino acid residue which has a similar side chain) amino acidresidues or nucleotides to a second amino acid or nucleotide sequencesuch that the first and second amino acid or nucleotide sequences sharecommon structural domains or motifs and/or a common functional activity.For example, amino acid or nucleotide sequences which share commonstructural domains have at least 30%, 40%, or 50% identity, preferably60% identity, more preferably 70%-80%, and even more preferably 90-95%identity across the amino acid sequences of the domains and contain atleast one and preferably two structural domains or motifs, are definedherein as sufficiently identical. Furthermore, amino acid or nucleotidesequences which share at least 30%, 40%, or 50%, preferably 60%, morepreferably 70-80%, or 90-95% identity and share a common functionalactivity are defined herein as sufficiently identical.

Preferred proteins are PCIP proteins having at least one calcium bindingdomain and, preferably, a PCIP activity. Other preferred proteins arePCIP proteins having at least one calcium binding domain, and are,preferably, encoded by a nucleic acid molecule having a nucleotidesequence which hybridizes under stringent hybridization conditions to anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, or 90% of the length of the referencesequence (e.g., when aligning a second sequence to the PCIP amino acidsequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ ID NO:72 having 177 amino acidresidues, at least 80, preferably at least 100, more preferably at least120, even more preferably at least 140, and even more preferably atleast 150, 160 or 170 amino acid residues are aligned). The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blosum 62 matrix or a PAM250 matrix,and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1,2, 3, 4, 5, or 6. In yet another preferred embodiment, the percentidentity between two nucleotide sequences is determined using the GAPprogram in the GCG software package (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search against publicdatabases to, for example, identify other family members or relatedsequences. Such searches can be performed using the NBLAST and XBLASTprograms (version 2.0) of Altschul, et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to PCIP nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to PCIP proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

The invention also provides PCIP chimeric or fusion proteins. As usedherein, a PCIP “chimeric protein” or “fusion protein” comprises a PCIPpolypeptide operatively linked to a non-PCIP polypeptide. An “PCIPpolypeptide” refers to a polypeptide having an amino acid sequencecorresponding to PCIP, whereas a “non-PCIP polypeptide” refers to apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the PCIP protein, e.g., aprotein which is different from the PCIP protein and which is derivedfrom the same or a different organism. Within a PCIP fusion protein thePCIP polypeptide can correspond to all or a portion of a PCIP protein.In a preferred embodiment, a PCIP fusion protein comprises at least onebiologically active portion of a PCIP protein. In another preferredembodiment, a PCIP fusion protein comprises at least two biologicallyactive portions of a PCIP protein. Within the fusion protein, the term“operatively linked” is intended to indicate that the PCIP polypeptideand the non-PCIP polypeptide are fused in-frame to each other. Thenon-PCIP polypeptide can be fused to the N-terminus or C-terminus of thePCIP polypeptide.

For example, in one embodiment, the fusion protein is a GST-PCIP fusionprotein in which the PCIP sequences are fused to the C-terminus of theGST sequences. Such fusion proteins can facilitate the purification ofrecombinant PCIP.

In another embodiment, the fusion protein is a PCIP protein containing aheterologous signal sequence at its N-terminus. In certain host cells(e.g., mammalian host cells), expression and/or secretion of PCIP can beincreased through use of a heterologous signal sequence.

The PCIP fusion proteins of the invention can be incorporated intopharmaceutical compositions and administered to a subject in vivo. ThePCIP fusion proteins can be used to affect the bioavailability of a PCIPsubstrate. Use of PCIP fusion proteins may be useful therapeutically forthe treatment of potassium channel associated disorders such as CNSdisorders, e.g., neurodegenerative disorders such as Alzheimer'sdisease, dementias related to Alzheimer's disease (such as Pick'sdisease), Parkinson's and other Lewy diffuse body diseases, multiplesclerosis, amyotrophic lateral sclerosis, progressive supranuclearpalsy, epilepsy and Jakob-Creutzfieldt disease; psychiatric disorders,e.g., depression, schizophrenic disorders, Korsakoffs psychosis, mania,anxiety disorders, or phobic disorders; learning or memory disorders,e.g., amnesia or age-related memory loss; and neurological disorders;e.g., migraine.

Moreover, the PCIP-fusion proteins of the invention can be used asimmunogens to produce anti-PCIP antibodies in a subject, to purify PCIPligands and in screening assays to identify molecules which inhibit theinteraction of PCIP with a PCIP substrate.

Preferably, a PCIP chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). APCIP-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the PCIP protein.

The present invention also pertains to variants of the PCIP proteinswhich function as either PCIP agonists (mimetics) or as PCIPantagonists. Variants of the PCIP proteins can be generated bymutagenesis, e.g., discrete point mutation or truncation of a PCIPprotein. An agonist of the PCIP proteins can retain substantially thesame, or a subset, of the biological activities of the naturallyoccurring form of a PCIP protein. An antagonist of a PCIP protein caninhibit one or more of the activities of the naturally occurring form ofthe PCIP protein by, for example, competitively modulating a potassiumchannel mediated activity of a PCIP protein. Thus, specific biologicaleffects can be elicited by treatment with a variant of limited function.In one embodiment, treatment of a subject with a variant having a subsetof the biological activities of the naturally occurring form of theprotein has fewer side effects in a subject relative to treatment withthe naturally occurring form of the PCIP protein.

In one embodiment, variants of a PCIP protein which function as eitherPCIP agonists (mimetics) or as PCIP antagonists can be identified byscreening combinatorial libraries of mutants, e.g., truncation mutants,of a PCIP protein for PCIP protein agonist or antagonist activity. Inone embodiment, a variegated library of PCIP variants is generated bycombinatorial mutagenesis at the nucleic acid level and is encoded by avariegated gene library. A variegated library of PCIP variants can beproduced by, for example, enzymatically ligating a mixture of syntheticoligonucleotides into gene sequences such that a degenerate set ofpotential PCIP sequences is expressible as individual polypeptides, oralternatively, as a set of larger fusion proteins (e.g., for phagedisplay) containing the set of PCIP sequences therein. There are avariety of methods which can be used to produce libraries of potentialPCIP variants from a degenerate oligonucleotide sequence. Chemicalsynthesis of a degenerate gene sequence can be performed in an automaticDNA synthesizer, and the synthetic gene then ligated into an appropriateexpression vector. Use of a degenerate set of genes allows for theprovision, in one mixture, of all of the sequences encoding the desiredset of potential PCIP sequences. Methods for synthesizing degenerateoligonucleotides are known in the art (see, e.g., Narang, S. A. (1983)Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323;Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic AcidRes. 11:477.

In addition, libraries of fragments of a PCIP protein coding sequencecan be used to generate a variegated population of PCIP fragments forscreening and subsequent selection of variants of a PCIP protein. In oneembodiment, a library of coding sequence fragments can be generated bytreating a double stranded PCR fragment of a PCIP coding sequence with anuclease under conditions wherein nicking occurs only about once permolecule, denaturing the double stranded DNA, renaturing the DNA to formdouble stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes. by treatment with S1 nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the PCIP protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of PCIP proteins. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recrusive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify PCIP variants (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci.USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering6(3):327-331).

In one embodiment, cell based assays can be exploited to analyze avariegated PCIP library. For example, a library of expression vectorscan be transfected into a cell line which ordinarily possesses apotassium channel mediated activity. The effect of the PCIP mutant onthe potassium channel mediated activity can then be detected, e.g., byany of a number of enzymatic assays or by detecting the release of aneurotransmitter. Plasmid DNA can then be recovered from the cells whichscore for inhibition, or alternatively, potentiation of the potassiumchannel mediated activity, and the individual clones furthercharacterized.

An isolated PCIP protein, or a portion or fragment thereof, can be usedas an immunogen to generate antibodies that bind PCIP using standardtechniques for polyclonal and monoclonal antibody preparation. Afull-length PCIP protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of PCIP for use as immunogens. Theantigenic peptide of PCIP comprises at least 8 amino acid residues ofthe amino acid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ IDNO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ IDNO:38, SEQ ID NO:40, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ IDNO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:70, or SEQ ID NO:72 andencompasses an epitope of PCIP such that an antibody raised against thepeptide forms a specific immune complex with PCIP. Preferably, theantigenic peptide comprises at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues.

Preferred epitopes encompassed by the antigenic peptide are regions ofPCIP that are located on the surface of the protein, e.g., hydrophilicregions, as well as regions with high antigenicity.

A PCIP immunogen typically is used to prepare antibodies by immunizing asuitable subject, (e.g., rabbit, goat, mouse or other mammal) with theimmunogen. An appropriate immunogenic preparation can contain, forexample, recombinantly expressed PCIP protein or a chemicallysynthesized PCIP polypeptide. The preparation can further include anadjuvant, such as Freund's complete or incomplete adjuvant, or similarimmunostimulatory agent. Immunization of a suitable subject with animmunogenic PCIP preparation induces a polyclonal anti-PCIP antibodyresponse.

Accordingly, another aspect of the invention pertains to anti-PCIPantibodies. The term “antibody” as used herein refers to immunoglobulinmolecules and immunologically active portions of immunoglobulinmolecules, i.e., molecules that contain an antigen binding site whichspecifically binds (immunoreacts with) an antigen, such as PCIP.Examples of immunologically active portions of immunoglobulin moleculesinclude F(ab) and F(ab′)₂ fragments which can be generated by treatingthe antibody with an enzyme such as pepsin. The invention providespolyclonal and monoclonal antibodies that bind PCIP. The term“monoclonal antibody” or “monoclonal antibody composition”, as usedherein, refers to a population of antibody molecules that contain onlyone species of an antigen binding site capable of immunoreacting with aparticular epitope of PCIP. A monoclonal antibody composition thustypically displays a single binding affinity for a particular PCIPprotein with which it immunoreacts.

Polyclonal anti-PCIP antibodies can be prepared as described above byimmunizing a suitable subject with a PCIP immunogen. The anti-PCIPantibody titer in the immunized subject can be monitored over time bystandard techniques, such as with an enzyme linked immunosorbent assay(ELISA) using immobilized PCIP. If desired, the antibody moleculesdirected against PCIP can be isolated from the mammal (e.g., from theblood) and further purified by well known techniques, such as protein Achromatography to obtain the IgG fraction. At an appropriate time afterimmunization, e.g., when the anti-PCIP antibody titers are highest,antibody-producing cells can be obtained from the subject and used toprepare monoclonal antibodies by standard techniques, such as thehybridoma technique originally described by Kohler and Milstein (1975)Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J. Biol. Chem .255:4980-83; Yeh et al.(1976) Proc. Natl. Acad. Sci. USA 76:2927-31; and Yeh et al. (1982) Int.J. Cancer 29:269-75), the more recent human B cell hybridoma technique(Kozbor et al. (1983) Immunol Today 4:72), the EBV-hybridoma technique(Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., pp. 77-96) or trioma techniques. The technology forproducing monoclonal antibody hybridomas is well known (see generally R.H. Kenneth, in Monoclonal Antibodies: A New Dimension In BiologicalAnalyses, Plenum Publishing Corp., New York, N.Y. (1980); E. A. Lerner(1981) Yale J. Biol. Med., 54:387-402; M. L. Gefter et al. (1977)Somatic Cell Genet. 3:231-36). Briefly, an immortal cell line (typicallya myeloma) is fused to lymphocytes (typically splenocytes) from a mammalimmunized with a PCIP immunogen as described above, and the culturesupernatants of the resulting hybridoma cells are screened to identify ahybridoma producing a monoclonal antibody that binds PCIP.

Any of the many well known protocols used for fusing lymphocytes andimmortalized cell lines can be applied for the purpose of generating ananti-PCIP monoclonal antibody (see, e.g., G. Galfre et al. (1977) Nature266:55052; Gefter et al. Somatic Cell Genet., cited supra; Lerner, YaleJ. Biol. Med., cited supra; Kenneth, Monoclonal Antibodies, citedsupra). Moreover, the ordinarily skilled worker will appreciate thatthere are many variations of such methods which also would be useful.Typically, the immortal cell line (e.g., a myeloma cell line) is derivedfrom the same mammalian species as the lymphocytes. For example, murinehybridomas can be made by fusing lymphocytes from a mouse immunized withan immunogenic preparation of the present invention with an immortalizedmouse cell line. Preferred immortal cell lines are mouse myeloma celllines that are sensitive to culture medium containing hypoxanthine,aminopterin and thymidine (“HAT medium”). Any of a number of myelomacell lines can be used as a fusion partner according to standardtechniques, e.g., the P3-NS 1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14myeloma lines. These myeloma lines are available from ATCC. Typically,HAT-sensitive mouse myeloma cells are fused to mouse splenocytes usingpolyethylene glycol (“PEG”). Hybridoma cells resulting from the fusionare then selected using HAT medium, which kills unfused andunproductively fused myeloma cells (unfused splenocytes die afterseveral days because they ate not transformed). Hybridoma cellsproducing a monoclonal antibody of the invention are detected byscreening the hybridoma culture supernatants for antibodies that bindPCIP, e.g., using a standard ELISA assay.

Alternative to preparing monoclonal antibody-secreting hybridomas, amonoclonal anti-PCIP antibody can be identified and isolated byscreening a recombinant combinatorial immunoglobulin library (e.g., anantibody phage display library) with PCIP to thereby isolateimmunoglobulin library members that bind PCIP. Kits for generating andscreening phage display libraries are commercially available (e.g., thePharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; andthe Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612).Additionally, examples of methods and reagents particularly amenable foruse in generating and screening antibody display library can be foundin, for example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCTInternational Publication No. WO 92/18619; Dower et al. PCTInternational Publication No. WO 91/17271; Winter et al. PCTInternational Publication WO 92/20791; Markland et al. PCT InternationalPublication No. WO 92/15679; Breitling et al. PCT InternationalPublication WO 93/01288; McCafferty et al. PCT International PublicationNo. WO 92/01047; Garrard et al. PCT International Publication No. WO92/09690; Ladner et al. PCT International Publication No. WO 90/02809;Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum.Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275-1281;Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol.Biol. 226:889-896; Clarkson et al. (1991) Nature 352:624-628; Gram etal. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc. Acid Res.19:4133-4137; Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982; and McCafferty et al. Nature (1990) 348:552-554.

Additionally, recombinant anti-PCIP antibodies, such as chimeric andhumanized monoclonal antibodies, comprising both human and non-humanportions, which can be made using standard recombinant DNA techniques,are within the scope of the invention. Such chimeric and humanizedmonoclonal antibodies can be produced by recombinant DNA techniquesknown in the art, for example using methods described in Robinson et al.International Application No. PCT/US86/02269; Akira, et al. EuropeanPatent Application 184,187; Taniguchi, M., European Patent Application171,496; Morrison et al. European Patent Application 173,494; Neubergeret al. PCT International Publication No. WO 86/01533; Cabilly et al.U.S. Pat. No. 4,816,567; Cabilly et al. European Patent Application125,023; Better et al. (1988) Science 240:1041-1043; Liu et al. (1987)Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987) J. Immunol.139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci. USA 84:214-218;Nishimura et al. (1987) Canc. Res. 47:999-1005; Wood et al. (1985)Nature 314:446-449; and Shaw et al. (1988) J. Natl. Cancer Inst.80:1553-1559); Morrison, S. L. (1985) Science 229:1202-1207; Oi et al.(1986) BioTechniques 4:214; Winter U.S. Pat. No. 5,225,539; Jones et al.(1986) Nature 321:552-525; Verhoeyan et al. (1988) Science 239:1534; andBeidler et al. (1988) J. Immunol. 141:4053-4060.

An anti-PCIP antibody (e.g., monoclonal antibody) can be used to isolatePCIP by standard techniques, such as affinity chromatography orimmunoprecipitation. An anti-PCIP antibody can facilitate thepurification of natural PCIP from cells and of recombinantly producedPCIP expressed in host cells. Moreover, an anti-PCIP antibody can beused to detect PCIP protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the PCIP protein. Anti-PCIP antibodies can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, -galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

III. Recombinant Expression Vectors and Host Cells

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing a nucleic acid encoding a PCIP protein(or a portion thereof). As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a “plasmid”, whichrefers to a circular double stranded DNA loop into which additional DNAsegments can be ligated. Another type of vector is a viral vector,wherein additional DNA segments can be ligated into the viral genome.Certain vectors are capable of autonomous replication in a host cellinto which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, which is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerwhich allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, and the like. The expressionvectors of the invention can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein (e.g., PCIP proteins,mutant forms of PCIP proteins, fusion proteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of PCIP proteins in prokaryotic or eukaryotic cells. Forexample, PCIP proteins can be expressed in bacterial cells such as E.coli, insect cells (using baculovirus expression vectors) yeast cells ormammalian cells. Suitable host cells are discussed further in Goeddel,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). Alternatively, the recombinant expressionvector can be transcribed and translated in vitro, for example using T7promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, in fusion expressionvectors, a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent topurification of the fusion protein. Such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin and enterokinase.Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith, D. B. and Johnson, K. S. (1988) Gene 67:31-40), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein.

Purified fusion proteins can be utilized in PCIP activity assays, (e.g.,direct assays or competitive assays described in detail below), or togenerate antibodies specific for PCIP proteins, for example. In apreferred embodiment, a PCIP fusion protein expressed in a retroviralexpression vector of the present invention can be utilized to infectbone marrow cells which are subsequently transplanted into irradiatedrecipients. The pathology of the subject recipient is then examinedafter sufficient time has passed (e.g., six (6) weeks).

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amann et al., (1988) Gene 69:301-315) and pET 11d (Studieret al., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 60-89). Target gene expression from thepTrc vector relies on host RNA polymerase transcription from a hybridtrp-lac fusion promoter. Target gene expression from the pET 11d vectorrelies on transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gnl). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a residentprophage harboring a T7 gnl gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein (Gottesman, S., GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence of the nucleic acid to be inserted into an expressionvector so that the individual codons for each amino acid are thosepreferentially utilized in E. coli (Wada et al., (1992) Nucleic AcidsRes. 20:2111-2118). Such alteration of nucleic acid sequences of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the PCIP expression vector is a yeast expressionvector. Examples of vectors for expression in yeast S. cerivisae includepYepSec1 (Baldari, et al., (1987) Embo J. 6:229-234), pMFa (Kurjan andHerskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ(InVitrogen Corp, San Diego, Calif.).

Alternatively, PCIP proteins can be expressed in insect cells usingbaculovirus expression vectors. Baculovirus vectors available forexpression of proteins in cultured insect cells (e.g., Sf9 cells)include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165)and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).

In yet another embodiment, a nucleic acid of the invention is expressedin mammalian cells using a mammalian expression vector. Examples ofmammalian expression vectors include pCDM8 (Seed, B. (1987) Nature329:840) and pMT2PC (Kaufman et al. (1987) EMBO J. 6:187-195). When usedin mammalian cells, the expression vector's control functions are oftenprovided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. For other suitable expression systems for bothprokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert et al.(1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame andEaton (1988) Adv. Immunol 43:235-275), in particular promoters of T cellreceptors (Winoto and Baltimore (1989) EMBO J 8:729-733) andimmunoglobulins (Banerji et al. (1983) Cell 33:729-740; Queen andBaltimore (1983) Cell 33:741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle (1989) Proc. Natl. Acad. Sci.USA 86:5473-5477), pancreas-specific promoters (Edlund et al. (1985)Science 230:912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, for example the murine hox promoters (Kessel and Gruss(1990) Science 249:374-379) and the α-fetoprotein promoter (Campes andTilghman (1989) Genes Dev. 3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. That is, the DNA molecule isoperatively linked to a regulatory sequence in a manner which allows forexpression (by transcription of the DNA molecule) of an RNA moleculewhich is antisense to PCIP mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosenwhich direct the continuous expression of the antisense RNA molecule ina variety of cell types, for instance viral promoters and/or enhancers,or regulatory sequences can be chosen which direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see Weintraub, H. et al., Antisense RNAas a molecular tool for genetic analysis, Reviews—Trends in Genetics,Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector of the invention has been introduced. Theterms “host cell” and “recombinant host cell” are used interchangeablyherein. It is understood that such terms refer not only to theparticular subject cell but to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aPCIP protein can be expressed in bacterial cells such as E. coli, insectcells, yeast or mammalian cells (such as Chinese hamster ovary cells(CHO) or COS cells). Other suitable host cells are known to thoseskilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest.Preferred selectable markers include those which confer resistance todrugs, such as G418, hygromycin and methotrexate. Nucleic acid encodinga selectable marker can be introduced into a host cell on the samevector as that encoding a PCIP protein or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell of the invention, such as a prokaryotic or eukaryotic hostcell in culture, can be used to produce (i.e., express) a PCIP protein.Accordingly, the invention further provides methods for producing a PCIPprotein using the host cells of the invention. In one embodiment, themethod comprises culturing the host cell of invention (into which arecombinant expression vector encoding a PCIP protein has beenintroduced) in a suitable medium such that a PCIP protein is produced.In another embodiment, the method further comprises isolating a PCIPprotein from the medium or the host cell.

The host cells of the invention can also be used to produce non-humantransgenic animals. For example, in one embodiment, a host cell of theinvention is a fertilized oocyte or an embryonic stem cell into whichPCIP-coding sequences have been introduced. Such host cells can then beused to create non-human transgenic animals in which exogenous PCIPsequences have been introduced into their genome or homologousrecombinant animals in which endogenous PCIP sequences have beenaltered. Such animals are useful for studying the function and/oractivity of a PCIP and for identifying and/or evaluating modulators ofPCIP activity. As used herein, a “transgenic animal” is a non-humananimal, preferably a mammal, more preferably a rodent such as a rat ormouse, in which one or more of the cells of the animal includes atransgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, etc. Atransgene is exogenous DNA which is integrated into the genome of a cellfrom which a transgenic animal develops and which remains in the genomeof the mature animal, thereby directing the expression of an encodedgene product in one or more cell types or tissues of the transgenicanimal. As used herein, a “homologous recombinant animal” is a non-humananimal, preferably a mammal, more preferably a mouse, in which anendogenous PCIP gene has been altered by homologous recombinationbetween the endogenous gene and an exogenous DNA molecule introducedinto a cell of the animal, e.g., an embryonic cell of the animal, priorto development of the animal.

A transgenic animal of the invention can be created by introducing aPCIP-encoding nucleic acid into the male pronuclei of a fertilizedoocyte, e.g., by microinjection, retroviral infection, and allowing theoocyte to develop in a pseudopregnant female foster animal. The PCIPcDNA sequence of SEQ ID NO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ IDNO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ IDNO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ IDNO:39, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ IDNO:71 can be introduced as a transgene into the genome of a non-humananimal. Alternatively, a nonhuman homologue of a human PCIP gene, suchas a mouse or rat PCIP gene, can be used as a transgene. Alternatively,a PCIP gene homologue, such as another PCIP family member, can beisolated based on hybridization to the PCIP cDNA sequences of SEQ IDNO:1, SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46,SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54,SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71 or the DNAinsert of the plasmid deposited with ATCC as Accession Number 98936,98937, 98938, 98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946,98947, 98948, 98949, 98950, 98951, 98991, 98993, or 98994 (describedfurther in subsection I above) and used as a transgene. Intronicsequences and polyadenylation signals can also be included in thetransgene to increase the efficiency of expression of the transgene. Atissue-specific regulatory sequence(s) can be operably linked to a PCIPtransgene to direct expression of a PCIP protein to particular cells.Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of a PCIP transgene in its genome and/or expression of PCIPmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene encoding a PCIPprotein can further be bred to other transgenic animals carrying othertransgenes.

To create a homologous recombinant animal, a vector is prepared whichcontains at least a portion of a PCIP gene into which a deletion,addition or substitution has been introduced to thereby alter, e.g.,functionally disrupt, the PCIP gene. The PCIP gene can be a human gene(e.g., the cDNA of SEQ ID NO:1), but more preferably, is a non-humanhomologue of a human PCIP gene (e.g., the cDNA of SEQ ID NO:3 or 5). Forexample, a mouse PCIP gene can be used to construct a homologousrecombination vector suitable for altering an endogenous PCIP gene inthe mouse genome. In a preferred embodiment, the vector is designed suchthat, upon homologous recombination, the endogenous PCIP gene isfunctionally disrupted (i.e., no longer encodes a functional protein;also referred to as a “knock out” vector). Alternatively, the vector canbe designed such that, upon homologous recombination, the endogenousPCIP gene is mutated or otherwise altered but still encodes functionalprotein (e.g., the upstream regulatory region can be altered to therebyalter the expression of the endogenous PCIP protein). In the homologousrecombination vector, the altered portion of the PCIP gene is flanked atits 5′ and 3′ ends by additional nucleic acid sequence of the PCIP geneto allow for homologous recombination to occur between the exogenousPCIP gene carried by the vector and an endogenous PCIP gene in anembryonic stem cell. The additional flanking PCIP nucleic acid sequenceis of sufficient length for successful homologous recombination with theendogenous gene. Typically, several kilobases of flanking DNA (both atthe 5′ and 3′ ends) are included in the vector (see e.g., Thomas, K. R.and Capecchi, M. R. (1987) Cell 51:503 for a description of homologousrecombination vectors). The vector is introduced into an embryonic stemcell line (e.g., by electroporation) and cells in which the introducedPCIP gene has homologously recombined with the endogenous PCIP gene areselected (see e.g., Li, E. et al. (1992) Cell 69:915). The selectedcells are then injected into a blastocyst of an animal (e.g., a mouse)to form aggregation chimeras (see e.g., Bradley, A. in Teratocarcinomasand Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed.(IRL, Oxford, 1987) pp. 113-152). A chimeric embryo can then beimplanted into a suitable pseudopregnant female foster animal and theembryo brought to term. Progeny harboring the homologously recombinedDNA in their germ cells can be used to breed animals in which all cellsof the animal contain the homologously recombined DNA by germlinetransmission of the transgene. Methods for constructing homologousrecombination vectors and homologous recombinant animals are describedfurther in Bradley, A. (1991) Current Opinion in Biotechnology 2:823-829and in PCT International Publication Nos.: WO 90/11354 by Le Mouellec etal.; WO 91/01140 by Smithies et al.; WO 92/0968 by Zijlstra et al.; andWO 93/04169 by Bems et al.

In another embodiment, transgenic non-humans animals can be producedwhich contain selected systems which allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. (1992) Proc. Natl. Acad Sci.USA 89:6232-6236. Another example of a recombinase system is the FLPrecombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991)Science 251:1351-1355. If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein are required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. (1997)Nature 385:810-813 and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G₀ phase. The quiescent cell can then be fused, e.g., throughthe use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated. Therecontructed oocyte is then cultured such that it develops to morula orblastocyte and then transferred to pseudopregnant female foster animal.The offspring borne of this female foster animal will be a clone of theanimal from which the cell, e.g., the somatic cell, is isolated.

IV. Pharmaceutical Compositions

The PCIP nucleic acid molecules, fragments of PCIP proteins, andanti-PCIP antibodies (also referred to herein as “active compounds”) ofthe invention can be incorporated into pharmaceutical compositionssuitable for administration. Such compositions typically comprise thenucleic acid molecule, protein, or antibody and a pharmaceuticallyacceptable carrier. As used herein the language “pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound (e.g., a fragment of a PCIP protein or an anti-PCIP antibody)in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or, preferably, can include aseries of treatments.

In a preferred example, a subject is treated with antibody, protein, orpolypeptide in the range of between about 0.1 to 20 mg/kg body weight,one time per week for between about 1 to 10 weeks, preferably between 2to 8 weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. It will also be appreciated thatthe effective dosage of antibody, protein, or polypeptide used fortreatment may increase or decrease over the course of a particulartreatment. Changes in dosage may result and become apparent from theresults of diagnostic assays as described herein.

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e,. including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds having a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds. It is understood that appropriate doses of smallmolecule agents depends upon a number of factors within the ken of theordinarily skilled physician, veterinarian, or researcher. The dose(s)of the small molecule will vary, for example, depending upon theidentity, size, and condition of the subject or sample being treated,further depending upon the route by which the composition is to beadministered, if applicable, and the effect which the practitionerdesires the small molecule to have upon the nucleic acid or polypeptideof the invention. Exemplary doses include milligram or microgram amountsof the small molecule per kilogram of subject or sample weight (e.g.,about 1 microgram per kilogram to about 500 milligrams per kilogram,about 100 micrograms per kilogram to about 5 milligrams per kilogram, orabout 1 microgram per kilogram to about 50 micrograms per kilogram. Itis furthermore understood that appropriate doses of a small moleculedepend upon the potency of the small molecule with respect to theexpression or activity to be modulated. Such appropriate doses may bedetermined using the assays described herein. When one or more of thesesmall molecules is to be administered to an animal (e.g., a human) inorder to modulate expression or activity of a polypeptide or nucleicacid of the invention, a physician, veterinarian, or researcher may, forexample, prescribe a relatively low dose at first, subsequentlyincreasing the dose until an appropriate response is obtained. Inaddition, it is understood that the specific dose level for anyparticular animal subject will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated.

Further, an antibody (or fragment thereof) may be conjugated to atherapeutic moiety such as a cytotoxin, a therapeutic agent or aradioactive metal ion. A cytotoxin or cytotoxic agent includes any agentthat is detrimental to cells. Examples include taxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carnustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, .alpha.-interferon, .beta.-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev., 62:119-58 (1982). Alternatively, an antibody can beconjugated to a second antibody to Form an antibody heteroconjugate asdescribed by Segal in U.S. Pat. No. 4,676,980.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

V. Uses and Methods of the Invention

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Asdescribed herein, a PCIP protein of the invention has one or more of thefollowing activities: (1) interaction with (e.g., bind to) a potassiumchannel protein or portion thereof; (2) regulation of thephosphorylation state of a potassium channel protein or portion thereof;(3) association with (e.g., binding to) calcium and can, for example,act as a calcium dependent kinase, e.g., phosphorylate a potassiumchannel or a G-protein coupled receptor in a calcium-dependent manner;(4) association with (e.g., binding to) calcium and can, for example,act as a calcium dependent transcription factor; (5) modulation of apotassium channel mediated activity in a cell (e.g., a neuronal cell)to, for example, beneficially affect the cell; (6) modulation ofchromatin formation in a cell, e.g., a neuronal cell; (7) modulation ofvesicular traffic and protein transport in a cell, e.g., a neuronalcell; (8) modulation of cytokine signaling in a cell, e.g., a neuronalcell; (9) regulation of the association of a potassium channel proteinor portion thereof with the cellular cytoskeleton; (10) modulation ofcellular proliferation; (11) modulation of the release ofneurotransmitters; (12) modulation of membrane excitability; (13)influencing of the resting potential of membranes; (14) modulation ofwave forms and frequencies of action potentials; and (15) modulation ofthresholds of excitation and, thus, can be used to, for example, (1)modulate the activity of a potassium channel protein or portion thereof;(2) modulate the phosphorylation state of a potassium channel protein orportion thereof; (3) modulate the phosphorylation state of a potassiumchannel or a G-protein coupled receptor in a calcium-dependent manner;(4) associate with (e.g., bind to) calcium and act as a calciumdependent transcription factor; (5) modulate a potassium channelmediated activity in a cell (e.g., a neuronal cell) to, for example,beneficially affect the cell; (6) modulate chromatin formation in acell, e.g., a neuronal cell; (7) modulate vesicular traffic and proteintransport in a cell, e.g., a neuronal cell; (8) modulate cytokinesignaling in a cell, e.g., a neuronal cell; (9) regulate the associationof a potassium channel protein or portion thereof with the cellularcytoskeleton; (10) modulate cellular proliferation; (11) modulate therelease of neurotransmitters; (12) modulate membrane excitability; (13)influence the resting potential of membranes; (14) modulate wave formsand frequencies of action potentials; and (15) modulate thresholds ofexcitation.

The isolated nucleic acid molecules of the invention can be used, forexample, to express PCIP protein (e.g., via a recombinant expressionvector in a host cell in gene therapy applications), to detect PCIP mRNA(e.g., in a biological sample) or a genetic alteration in a PCIP gene,and to modulate PCIP activity, as described further below. The PCIPproteins can be used to treat disorders characterized by insufficient orexcessive production of a PCIP substrate or production of PCIPinhibitors. In addition, the PCIP proteins can be used to screen fornaturally occurring PCIP substrates, to screen for drugs or compoundswhich modulate PCIP activity, as well as to treat disorderscharacterized by insufficient or excessive production of PCIP protein orproduction of PCIP protein forms which have decreased or aberrantactivity compared to PCIP wild type protein (e.g., CNS disorders such asneurodegenerative disorders, e.g., Alzheimer's disease, dementiasrelated to Alzheimer's disease (such as Pick's disease), Parkinson's andother Lewy diffuse body diseases, multiple sclerosis, amyotrophiclateral sclerosis, progressive supranuclear palsy, epilepsy andJakob-Creutzfieldt disease; psychiatric disorders, e.g., depression,schizophrenic disorders, Korsakoff's psychosis, mania, anxietydisorders, bipolar affective disorders, or phobic disorders; learning ormemory disorders, e.g., amnesia or age-related memory loss; neurologicaldisorders, e.g., migraine; pain disorders, e.g., hyperalgesia or painassociated with muscoloskeletal disorders; spinal cord injury; stroke;and head trauma). Moreover, the anti-PCIP antibodies of the inventioncan be used to detect and isolate PCIP proteins, regulate thebioavailability of PCIP proteins, and modulate PCIP activity.

A. Screening Assays

The invention provides a method (also referred to herein as a “screeningassay”) for identifying modulators, i.e., candidate or test compounds oragents (e.g., peptides, peptidomimetics, small molecules or other drugs)which bind to PCIP proteins, have a stimulatory or inhibitory effect on,for example, PCIP expression or PCIP activity, or have a stimulatory orinhibitory effect on, for example, the expression or activity of PCIPsubstrate.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of a PCIP protein or polypeptideor biologically active portion thereof. In another embodiment, theinvention provides assays for screening candidate or test compoundswhich bind to or modulate the activity of a PCIP protein or polypeptideor biologically active portion thereof. The test compounds of thepresent invention can be obtained using any of the numerous approachesin combinatorial library methods known in the art, including: biologicallibraries; spatially addressable parallel solid phase or solution phaselibraries; synthetic library methods requiring deconvolution; the‘one-bead one-compound’ library method; and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds (Lam, K. S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a PCIP protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tomodulate PCIP activity, e.g., binding to a potassium channel or aportion thereof, is determined. Determining the ability of the testcompound to modulate PCIP activity can be accomplished by monitoring,for example, the release of a neurotransmitter, e.g., dopamine, form acell which expresses PCIP such as a neuronal cell, e.g., a substantianigra neuronal cell. The cell, for example, can be of mammalian origin.Determining the ability of the test compound to modulate the ability ofPCIP to bind to a substrate can be accomplished, for example, bycoupling the PCIP substrate with a radioisotope or enzymatic label suchthat binding of the PCIP substrate to PCIP can be determined bydetecting the labeled PCIP substrate in a complex. For example,compounds (e.g., PCIP substrates) can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or³H, either directly or indirectly, and the radioisotope detected bydirect counting of radioemmission or by scintillation counting.Alternatively, compounds can be enzymatically labeled with, for example,horseradish peroxidase, alkaline phosphatase, or luciferase, and theenzymatic label detected by determination of conversion of anappropriate substrate to product.

It is also within the scope of this invention to determine the abilityof a compound (e.g., PCIP substrate) to interact with PCIP without thelabeling of any of the interactants. For example, a microphysiometer canbe used to detect the interaction of a compound with PCIP without thelabeling of either the compound or the PCIP. McConnell, H. M. et al.(1992) Science 257:1906-1912. As used herein, a “microphysiometer”(e.g., Cytosensor) is an analytical instrument that measures the rate atwhich a cell acidifies its environment using a light-addressablepotentiometric sensor (LAPS). Changes in this acidification rate can beused as an indicator of the interaction between a compound and PCIP.

In another embodiment, an assay is a cell-based assay comprisingcontacting a cell expressing a PCIP target molecule (e.g., a potassiumchannel or a fragment thereof) with a test compound and determining theability of the test compound to modulate (e.g. stimulate or inhibit) theactivity of the PCIP target molecule. Determining the ability of thetest compound to modulate the activity of a PCIP target molecule can beaccomplished, for example, by determining the ability of the PCIPprotein to bind to or interact with the PCIP target molecule, e.g., apotassium channel or a fragment thereof.

Determining the ability of the PCIP protein or a biologically activefragment thereof, to bind to or interact with a PCIP target molecule canbe accomplished by one of the methods described above for determiningdirect binding. In a preferred embodiment, determining the ability ofthe PCIP protein to bind to or interact with a PCIP target molecule canbe accomplished by determining the activity of the target molecule. Forexample, the activity of the target molecule can be determined bydetecting induction of a cellular second messenger of the target (i.e.,intracellular Ca²⁺, diacylglycerol, IP₃, and the like), detectingcatalytic/enzymatic activity of the target an appropriate substrate,detecting the induction of a reporter gene (comprising atarget-responsive regulatory element operatively linked to a nucleicacid encoding a detectable marker, e.g., luciferase), or detecting atarget-regulated cellular response such as the release of aneurotransmitter.

In yet another embodiment, an assay of the present invention is acell-free assay in which a PCIP protein or biologically active portionthereof is contacted with a test compound and the ability of the testcompound to bind to the PCIP protein or biologically active portionthereof is determined. Preferred biologically active portions of thePCIP proteins to be used in assays of the present invention includefragments which participate in interactions with non-PCIP molecules,e.g., potassium channels or fragments thereof, or fragments with highsurface probability scores. Binding of the test compound to the PCIPprotein can be determined either directly or indirectly as describedabove. In a preferred embodiment, the assay includes contacting the PCIPprotein or biologically active portion thereof with a known compoundwhich binds PCIP to form an assay mixture, contacting the assay mixturewith a test compound, and determining the ability of the test compoundto interact with a PCIP protein, wherein determining the ability of thetest compound to interact with a PCIP protein comprises determining theability of the test compound to preferentially bind to PCIP orbiologically active portion thereof as compared to the known compound.

In another embodiment, the assay is a cell-free assay in which a PCIPprotein or biologically active portion thereof is contacted with a testcompound and the ability of the test compound to modulate (e.g.,stimulate or inhibit) the activity of the PCIP protein or biologicallyactive portion thereof is determined. Determining the ability of thetest compound to modulate the activity of a PCIP protein can beaccomplished, for example, by determining the ability of the PCIPprotein to bind to a PCIP target molecule by one of the methodsdescribed above for determining direct binding. Determining the abilityof the PCIP protein to bind to a PCIP target molecule can also beaccomplished using a technology such as real-time BiomolecularInteraction Analysis (BIA). Sjolander, S. and Urbaniczky, C. (1991)Anal. Chem. 63:2338-2345 and Szabo et al. (1995) Curr. Opin. Struct.Biol. 5:699-705. As used herein, “BIA” is a technology for studyingbiospecific interactions in real time, without labeling any of theinteractants (e.g., BIAcore). Changes in the optical phenomenon ofsurface plasmon resonance (SPR) can be used as an indication ofreal-time reactions between biological molecules.

In an alternative embodiment, determining the ability of the testcompound to modulate the activity of a PCIP protein can be accomplishedby determining the ability of the PCIP protein to further modulate theactivity of a downstream effector of a PCIP target molecule. Forexample, the activity of the effector molecule on an appropriate targetcan be determined or the binding of the effector to an appropriatetarget can be determined as previously described.

In yet another embodiment, the cell-free assay involves contacting aPCIP protein or biologically active portion thereof with a knowncompound which binds the PCIP protein to form an assay mixture,contacting the assay mixture with a test compound, and determining theability of the test compound to interact with the PCIP protein, whereindetermining the ability of the test compound to interact with the PCIPprotein comprises determining the ability of the PCIP protein topreferentially bind to or modulate the activity of a PCIP targetmolecule.

The cell-free assays of the present invention are amenable to use ofboth soluble and/or membrane-bound forms of isolated proteins. In thecase of cell-free assays in which a membrane-bound form of an isolatedprotein is used (e.g., a potassium channel) it may be desirable toutilize a solubilizing agent such that the membrane-bound form of theisolated protein is maintained in solution. Examples of suchsolubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.

In more than one embodiment of the above assay methods of the presentinvention, it may be desirable to immobilize either PCIP or its targetmolecule to facilitate separation of complexed from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay. Binding of a test compound to a PCIP protein, or interaction of aPCIP protein with a target molecule in the presence and absence of acandidate compound, can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows one orboth of the proteins to be bound to a matrix. For example,glutathione-S-transferase/PCIP fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or PCIP protein, and the mixture incubated underconditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotitre plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of PCIPbinding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either a PCIPprotein or a PCIP target molecule can be immobilized utilizingconjugation of biotin and streptavidin. Biotinylated PCIP protein ortarget molecules can be prepared from biotin-NHS (N-hydroxy-succinimide)using techniques known in the art (e.g., biotinylation kit, PierceChemicals, Rockford, Ill.), and immobilized in the wells ofstreptavidin-coated 96 well plates (Pierce Chemical). Alternatively,antibodies reactive with PCIP protein or target molecules but which donot interfere with binding of the PCIP protein to its target moleculecan be derivatized to the wells of the plate, and unbound target or PCIPprotein trapped in the wells by antibody conjugation. Methods fordetecting such complexes, in addition to those described above for theGST-immobilized complexes, include immunodetection of complexes usingantibodies reactive with the PCIP protein or target molecule, as well asenzyme-linked assays which rely on detecting an enzymatic activityassociated with the PCIP protein or target molecule.

In a preferred embodiment, candidate or test compounds or agents aretested for their ability to inhibit or stimulate a PCIP molecule'sability to modulate vesicular traffic and protein transport in a cell,e.g., a neuronal cell, using the assays described in, for example,Komada M. et al. (1999) Genes Dev. 13(11):1475-85, and Roth M. G. et al.(1999) Chem. Phys. Lipids. 98(1-2):141-52, the contents of which areincorporated herein by reference.

In another preferred embodiment, candidate or test compounds or agentsare tested for their ability to inhibit or stimulate a PCIP molecule'sability to regulate the phosphorylation state of a potassium channelprotein or portion thereof, using for example, an in vitro kinase assay.Briefly, a PCIP target molecule, e.g., an immunoprecipitated potassiumchannel from a cell line expressing such a molecule, can be incubatedwith the PCIP protein and radioactive ATP, e.g., [γ-³²P] ATP, in abuffer containing MgCl₂ and MnCl₂, e.g., 10 mM MgCl₂ and 5 mM MnCl₂.Following the incubation, the immunoprecipitated PCIP target molecule,e.g., the potassium channel, can be separated by SDS-polyacrylamide gelelectrophoresis under reducing conditions, transferred to a membrane,e.g., a PVDF membrane, and autoradiographed. The appearance ofdetectable bands on the autoradiograph indicates that the PCIPsubstrate, e.g., the potassium channel, has been phosphorylated.Phosphoaminoacid analysis of the phosphorylated substrate can also beperformed in order to determine which residues on the PCIP substrate arephosphorylated. Briefly, the radiophosphorylated protein band can beexcised from the SDS gel and subjected to partial acid hydrolysis. Theproducts can then be separated by one-dimensional electrophoresis andanalyzed on, for example, a phosphoimager and compared toninhydrin-stained phosphoaminoacid standards. Assays such as thosedescribed in, for example, Tamaskovic R. et al. (1999) Biol. Chem.380(5):569-78, the contents of which are incorporated herein byreference, can also be used.

In another preferred embodiment, candidate or test compounds or agentsare tested for their ability to inhibit or stimulate a PCIP molecule'sability to associate with (e.g., bind) calcium, using for example, theassays described in Liu L. (1999) Cell Signal. 11(5):317-24 and Kawai T.et al. (1999) Oncogene 18(23):3471-80, the contents of which areincorporated herein by reference.

In another preferred embodiment, candidate or test compounds or agentsare tested for their ability to inhibit or stimulate a PCIP molecule'sability to modulate chromatin formation in a cell, using for example,the assays described in Okuwaki M. et al. (1998) J. Biol. Chem.273(51):34511-8 and Miyaji-Yamaguchi M. (1999) J. Mol. Biol. 290(2):547-557, the contents of which are incorporated herein by reference.

In yet another preferred embodiment, candidate or test compounds oragents are tested for their ability to inhibit or stimulate a PCIPmolecule's ability to modulate cellular proliferation, using forexample, the assays described in Baker F. L. et al. (1995) Cell Prolif.28(1):1-15, Cheviron N. et al. (1996) Cell Prolif. 29(8):437-46, Hu Z.W. et al. (1999) J. Pharmacol. Exp. Ther. 290(1):28-37 and Elliott K. etal. (1999) Oncogene 18(24):3564-73, the contents of which areincorporated herein by reference.

In a preferred embodiment, candidate or test compounds or agents aretested for their ability to inhibit or stimulate a PCIP molecule'sability to regulate the association of a potassium channel protein orportion thereof with the cellular cytoskeleton, using for example, theassays described in Gonzalez C. et al. (1998) Cell Mol. Biol.44(7):1117-27 and Chia C. P. et al. (1998) Exp. Cell Res. 244(1):340-8,the contents of which are incorporated herein by reference.

In another preferred embodiment, candidate or test compounds or agentsare tested for their ability to inhibit or stimulate a PCIP molecule'sability to modulate membrane excitability, using for example, the assaysdescribed in Bar-Sagi D. et al. (1985) J. Biol. Chem. 260(8):4740-4 andBarker J. L. et al. (1984) Neurosci. Lett. 47(3):313-8, the contents ofwhich are incorporated herein by reference.

In another preferred embodiment, candidate or test compounds or agentsare tested for their ability to inhibit or stimulate a PCIP molecule'sability to modulate cytokine signaling in a cell, e.g., a neuronal cell,the assays described in Nakashima Y. et al. (1999) J. Bone Joint Surg.Am. 81(5):603-15, the contents of which are incorporated herein byreference.

In another embodiment, modulators of PCIP expression are identified in amethod wherein a cell is contacted with a candidate compound and theexpression of PCIP mRNA or protein in the cell is determined. The levelof expression of PCIP mRNA or protein in the presence of the candidatecompound is compared to the level of expression of PCIP mRNA or proteinin the absence of the candidate compound. The candidate compound canthen be identified as a modulator of PCIP expression based on thiscomparison. For example, when expression of PCIP mRNA or protein isgreater (statistically significantly greater) in the presence of thecandidate compound than in its absence, the candidate compound isidentified as a stimulator of PCIP mRNA or protein expression.Alternatively, when expression of PCIP mRNA or protein is less(statistically significantly less) in the presence of the candidatecompound than in its absence, the candidate compound is identified as aninhibitor of PCIP mRNA or protein expression. The level of PCIP mRNA orprotein expression in the cells can be determined by methods describedherein for detecting PCIP mRNA or protein.

In yet another aspect of the invention, the PCIP proteins can be used as“bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura etal. (1993) J. Biol. Chem. 268:12046-12054; Bartel et al. (1993)Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with PCIP (“PCIP-binding proteins” or “PCIP-bp”) and areinvolved in PCIP activity (described in more detail in the Examplessection below). Such PCIP-binding proteins are also likely to beinvolved in the propagation of signals by the PCIP proteins or PCIPtargets as, for example, downstream elements of a PCIP-mediatedsignaling pathway. Alternatively, such PCIP-binding proteins are likelyto be PCIP inhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a PCIP protein isfused to a gene encoding the DNA binding domain of a known transcriptionfactor (e.g., GAL-4). In the other construct, a DNA sequence, from alibrary of DNA sequences, that encodes an unidentified protein (“prey”or “sample”) is fused to a gene that codes for the activation domain ofthe known transcription factor. If the “bait” and the “prey” proteinsare able to interact, in vivo, forming a PCIP-dependent complex, theDNA-binding and activation domains of the transcription factor arebrought into close proximity. This proximity allows transcription of areporter gene (e.g., LacZ) which is operably linked to a transcriptionalregulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing thefunctional transcription factor can be isolated and used to obtain thecloned gene which encodes the protein which interacts with the PCIPprotein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a PCIP modulating agent, an antisense PCIPnucleic acid molecule, a PCIP-specific antibody, or a PCIP-bindingpartner) can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal model to determine the mechanism of action of such an agent.Furthermore, this invention pertains to uses of novel agents identifiedby the above-described screening assays for treatments, e.g., treatmentsof a CNS disorder, as described herein.

B. Detection Assays

Portions or fragments of the cDNA sequences identified herein (and thecorresponding complete gene sequences) can be used in numerous ways aspolynucleotide reagents. For example, these sequences can be used to:(i) map their respective genes on a chromosome; and, thus, locate generegions associated with genetic disease; (ii) identify an individualfrom a minute biological sample (tissue typing); and (iii) aid inforensic identification of a biological sample. These applications aredescribed in the subsections below.

1. Chromosome Mapping

Once the sequence (or a portion of the sequence) of a gene has beenisolated, this sequence can be used to map the location of the gene on achromosome. This process is called chromosome mapping. Accordingly,portions or fragments of the PCIP nucleotide sequences, describedherein, can be used to map the location of the PCIP genes on achromosome. The mapping of the PCIP sequences to chromosomes is animportant first step in correlating these sequences with genesassociated with disease.

Briefly, PCIP genes can be mapped to chromosomes by preparing PCRprimers (preferably 15-25 bp in length) from the PCIP nucleotidesequences. Computer analysis of the PCIP sequences can be used topredict primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers can then beused for PCR screening of somatic cell hybrids containing individualhuman chromosomes. Only those hybrids containing the human genecorresponding to the PCIP sequences will yield an amplified fragment.

Somatic cell hybrids are prepared by fusing somatic cells from differentmammals (e.g., human and mouse cells). As hybrids of human and mousecells grow and divide, they gradually lose human chromosomes in randomorder, but retain the mouse chromosomes. By using media in which mousecells cannot grow, because they lack a particular enzyme, but humancells can, the one human chromosome that contains the gene encoding theneeded enzyme, will be retained. By using various media, panels ofhybrid cell lines can be established. Each cell line in a panel containseither a single human chromosome or a small number of human chromosomes,and a full set of mouse chromosomes, allowing easy mapping of individualgenes to specific human chromosomes. (D'Eustachio P. et al. (1983)Science 220:919-924). Somatic cell hybrids containing only fragments ofhuman chromosomes can also be produced by using human chromosomes withtranslocations and deletions.

PCR mapping of somatic cell hybrids is a rapid procedure for assigning aparticular sequence to a particular chromosome. Three or more sequencescan be assigned per day using a single thermal cycler. Using the PCIPnucleotide sequences to design oligonucleotide primers, sublocalizationcan be achieved with panels of fragments from specific chromosomes.Other mapping strategies which can similarly be used to map a PCIPsequence to its chromosome include in situ hybridization (described inFan, Y. et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27),pre-screening with labeled flow-sorted chromosomes, and pre-selection byhybridization to chromosome specific cDNA libraries.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. Chromosome spreads can be made usingcells whose division has been blocked in metaphase by a chemical such ascolcemid that disrupts the mitotic spindle. The chromosomes can betreated briefly with trypsin, and then stained with Giemsa. A pattern oflight and dark bands develops on each chromosome, so that thechromosomes can be identified individually. The FISH technique can beused with a DNA sequence as short as 500 or 600 bases. However, cloneslarger than 1,000 bases have a higher likelihood of binding to a uniquechromosomal location with sufficient signal intensity for simpledetection. Preferably 1,000 bases, and more preferably 2,000 bases willsuffice to get good results at a reasonable amount of time. For a reviewof this technique, see Verma et al., Human Chromosomes: A Manual ofBasic Techniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al. (1987)Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the PCIP gene, can bedetermined. If a mutation is observed in some or all of the affectedindividuals but not in any unaffected individuals, then the mutation islikely to be the causative agent of the particular disease. Comparisonof affected and unaffected individuals generally involves first lookingfor structural alterations in the chromosomes, such as deletions ortranslocations that are visible from chromosome spreads or detectableusing PCR based on that DNA sequence. Ultimately, complete sequencing ofgenes from several individuals can be performed to confirm the presenceof a mutation and to distinguish mutations from polymorphisms.

2. Tissue Typing

The PCIP sequences of the present invention can also be used to identifyindividuals from minute biological samples. The United States military,for example, is considering the use of restriction fragment lengthpolymorphism (RFLP) for identification of its personnel. In thistechnique, an individual's genomic DNA is digested with one or morerestriction enzymes, and probed on a Southern blot to yield unique bandsfor identification. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The sequences of the presentinvention are useful as additional DNA markers for RFLP (described inU.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can be used toprovide an alternative technique which determines the actualbase-by-base DNA sequence of selected portions of an individual'sgenome. Thus, the PCIP nucleotide sequences described herein can be usedto prepare two PCR primers from the 5′ and 3′ ends of the sequences.These primers can then be used to amplify an individual's DNA andsubsequently sequence it.

Panels of corresponding DNA sequences from individuals, prepared in thismanner, can provide unique individual identifications, as eachindividual will have a unique set of such DNA sequences due to allelicdifferences. The sequences of the present invention can be used toobtain such identification sequences from individuals and from tissue.The PCIP nucleotide sequences of the invention uniquely representportions of the human genome. Allelic variation occurs to some degree inthe coding regions of these sequences, and to a greater degree in thenoncoding regions. It is estimated that allelic variation betweenindividual humans occurs with a frequency of about once per each 500bases. Each of the sequences described herein can, to some degree, beused as a standard against which DNA from an individual can be comparedfor identification purposes. Because greater numbers of polymorphismsoccur in the noncoding regions, fewer sequences are necessary todifferentiate individuals. Non-coding sequences can comfortably providepositive individual identification with a panel of perhaps 10 to 1,000primers which each yield a noncoding amplified sequence of 100 bases. Ifpredicted coding sequences are used, a more appropriate number ofprimers for positive individual identification would be 500-2,000.

If a panel of reagents from PCIP nucleotide sequences described hereinis used to generate a unique identification database for an individual,those same reagents can later be used to identify tissue from thatindividual. Using the unique identification database, positiveidentification of the individual, living or dead, can be made fromextremely small tissue samples.

3. Use of Partial PCIP Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. Forensic biology is a scientific field employing genetic typingof biological evidence found at a crime scene as a means for positivelyidentifying, for example, a perpetrator of a crime. To make such anidentification, PCR technology can be used to amplify DNA sequencestaken from very small biological samples such as tissues, e.g., hair orskin, or body fluids, e.g., blood, saliva, or semen found at a crimescene. The amplified sequence can then be compared to a standard,thereby allowing identification of the origin of the biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions are particularly appropriate for this useas greater numbers of polymorphisms occur in the noncoding regions,making it easier to differentiate individuals using this technique.Examples of polynucleotide reagents include the PCIP nucleotidesequences or portions thereof, having a length of at least 20 bases,preferably at least 30 bases.

The PCIP nucleotide sequences described herein can further be used toprovide polynucleotide reagents, e.g., labeled or labelable probes whichcan be used in, for example, an in situ hybridization technique, toidentify a specific tissue, e.g., brain tissue. This can be very usefulin cases where a forensic pathologist is presented with a tissue ofunknown origin. Panels of such PCIP probes can be used to identifytissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., PCIP primers or probes canbe used to screen tissue culture for contamination (i.e. screen for thepresence of a mixture of different types of cells in a culture).

C. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual prophylactically. Accordingly, one aspect of the presentinvention relates to diagnostic assays for determining PCIP proteinand/or nucleic acid expression as well as PCIP activity, in the contextof a biological sample (e.g., blood, serum, cells, tissue) to therebydetermine whether an individual is afflicted with a disease or disorder,or is at risk of developing a disorder, associated with aberrant PCIPexpression or activity. The invention also provides for prognostic (orpredictive) assays for determining whether an individual is at risk ofdeveloping a disorder associated with PCIP protein, nucleic acidexpression or activity. For example, mutations in a PCIP gene can beassayed in a biological sample. Such assays can be used for prognosticor predictive purpose to thereby phophylactically treat an individualprior to the onset of a disorder characterized by or associated withPCIP protein, nucleic acid expression or activity.

Another aspect of the invention pertains to monitoring the influence ofagents (e.g., drugs, compounds) on the expression or activity of PCIP inclinical trials.

These and other agents are described in further detail in the followingsections.

1. Diagnostic Assays

An exemplary method for detecting the presence or absence of PCIPprotein or nucleic acid in a biological sample involves obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting PCIP protein ornucleic acid (e.g., mRNA, genomic DNA) that encodes PCIP protein suchthat the presence of PCIP protein or nucleic acid is detected in thebiological sample. A preferred agent for detecting PCIP mRNA or genomicDNA is a labeled nucleic acid probe capable of hybridizing to PCIP mRNAor genomic DNA. The nucleic acid probe can be, for example, afull-length PCIP nucleic acid, such as the nucleic acid of SEQ ID NO:1,SEQ ID NO:3 SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ IDNO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:69, or SEQ ID NO:71, or the DNA insert ofthe plasmid deposited with ATCC as Accession Number 98936, 98937, 98938,98939, 98940, 98941, 98942, 98943, 98944, 98945, 98946, 98947, 98948,98949, 98950, 98951, 98991, 98993, or 98994, or a portion thereof, suchas an oligonucleotide of at least 15, 30, 50, 100, 250 or 500nucleotides in length and sufficient to specifically hybridize understringent conditions to PCIP mRNA or genomic DNA. Other suitable probesfor use in the diagnostic assays of the invention are described herein.

A preferred agent for detecting PCIP protein is an antibody capable ofbinding to PCIP protein, preferably an antibody with a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity withanother reagent that is directly labeled. Examples of indirect labelinginclude detection of a primary antibody using a fluorescently labeledsecondary antibody and end-labeling of a DNA probe with biotin such thatit can be detected with fluorescently labeled streptavidin. The term“biological sample” is intended to include tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. That is, the detection method of the inventioncan be used to detect PCIP mRNA, protein, or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of PCIP mRNA include Northern hybridizations and in situhybridizations. In vitro techniques for detection of PCIP proteininclude enzyme linked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence. In vitro techniques fordetection of PCIP genomic DNA include Southern hybridizations.Furthermore, in vivo techniques for detection of PCIP protein includeintroducing into a subject a labeled anti-PCIP antibody. For example,the antibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample contains protein molecules fromthe test subject. Alternatively, the biological sample can contain mRNAmolecules from the test subject or genomic DNA molecules from the testsubject. A preferred biological sample is a serum sample isolated byconventional means from a subject.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting PCIP protein, mRNA, orgenomic DNA, such that the presence of PCIP protein, mRNA or genomic DNAis detected in the biological sample, and comparing the presence of PCIPprotein, mRNA or genomic DNA in the control sample with the presence ofPCIP protein, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting the presence of PCIPin a biological sample. For example, the kit can comprise a labeledcompound or agent capable of detecting PCIP protein or mRNA in abiological sample; means for determining the amount of PCIP in thesample; and means for comparing the amount of PCIP in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectPCIP protein or nucleic acid.

2. Prognostic Assays

The diagnostic methods described herein can furthermore be utilized toidentify subjects having or at risk of developing a disease or disorderassociated with aberrant PCIP expression or activity. For example, theassays described herein, such as the preceding diagnostic assays or thefollowing assays, can be utilized to identify a subject having or atrisk of developing a disorder associated with a misregulation in PCIPprotein activity or nucleic acid expression, such as a neurodegenerativedisorder, e.g., Alzheimer's disease, dementias related to Alzheimer'sdisease (such as Pick's disease), Parkinson's and other Lewy diffusebody diseases, multiple sclerosis, amyotrophic lateral sclerosis,progressive supranuclear palsy, epilepsy, and Jakob-Creutzfieldtdisease; a psychiatric disorder, e.g., depression, schizophrenicdisorders, Korsakoff's psychosis, mania, anxiety disorders, bipolaraffective disorders, or phobic disorders; a learning or memory disorder,e.g., amnesia or age-related memory loss; a neurological disorder, e.g.,migraine; a pain disorder, e.g., hyperalgesia or pain associated withmuscoloskeletal disorders; spinal cord injury; stroke; and head trauma.

Alternatively, the prognostic assays can be utilized to identify asubject having or at risk for developing a disorder associated with amisregulation in PCIP protein activity or nucleic acid expression, suchas a potassium channel associated disorder. Thus, the present inventionprovides a method for identifying a disease or disorder associated withaberrant PCIP expression or activity in which a test sample is obtainedfrom a subject and PCIP protein or nucleic acid (e.g., mRNA or genomicDNA) is detected, wherein the presence of PCIP protein or nucleic acidis diagnostic for a subject having or at risk of developing a disease ordisorder associated with aberrant PCIP expression or activity. As usedherein, a “test sample” refers to a biological sample obtained from asubject of interest. For example, a test sample can be a biologicalfluid (e.g., serum), cell sample, or tissue.

Furthermore, the prognostic assays described herein can be used todetermine whether a subject can be administered an agent (e.g., anagonist, antagonist, peptidomimetic, protein, peptide, nucleic acid,small molecule, or other drug candidate) to treat a disease or disorderassociated with aberrant PCIP expression or activity. For example, suchmethods can be used to determine whether a subject can be effectivelytreated with an agent for a CNS disorder. Thus, the present inventionprovides methods for determining whether a subject can be effectivelytreated with an agent for a disorder associated with aberrant PCIPexpression or activity in which a test sample is obtained and PCIPprotein or nucleic acid expression or activity is detected (e.g.,wherein the abundance of PCIP protein or nucleic acid expression oractivity is diagnostic for a subject that can be administered the agentto treat a disorder associated with aberrant PCIP expression oractivity).

The methods of the invention can also be used to detect geneticalterations in a PCIP gene, thereby determining if a subject with thealtered gene is at risk for a disorder characterized by misregulation inPCIP protein activity or nucleic acid expression, such as a CNSdisorder. In preferred embodiments, the methods include detecting, in asample of cells from the subject, the presence or absence of a geneticalteration characterized by at least one of an alteration affecting theintegrity of a gene encoding a PCIP-protein, or the mis-expression ofthe PCIP gene. For example, such genetic alterations can be detected byascertaining the existence of at least one of 1) a deletion of one ormore nucleotides from a PCIP gene; 2) an addition of one or morenucleotides to a PCIP gene; 3) a substitution of one or more nucleotidesof a PCIP gene, 4) a chromosomal rearrangement of a PCIP gene; 5) analteration in the level of a messenger RNA transcript of a PCIP gene, 6)aberrant modification of a PCIP gene, such as of the methylation patternof the genomic DNA, 7) the presence of a non-wild type splicing patternof a messenger RNA transcript of a PCIP gene, 8) a non-wild type levelof a PCIP-protein, 9) allelic loss of a PCIP gene, and 10) inappropriatepost-translational modification of a PCIP-protein. As described herein,there are a large number of assays known in the art which can be usedfor detecting alterations in a PCIP gene. A preferred biological sampleis a tissue or serum sample isolated by conventional means from asubject.

In certain embodiments, detection of the alteration involves the use ofa probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) Proc.Natl. Acad. Sci. USA 91:360-364), the latter of which can beparticularly useful for detecting point mutations in the PCIP-gene (seeAbravaya et al. (1995) Nucleic Acids Res .23:675-682). This method caninclude the steps of collecting a sample of cells from a subject,isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a PCIP gene under conditions such thathybridization and amplification of the PCIP-gene (if present) occurs,and detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al. (1988) Bio-Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In an alternative embodiment, mutations in a PCIP gene from a samplecell can be identified by alterations in restriction enzyme cleavagepatterns. For example, sample and control DNA is isolated, amplified(optionally), digested with one or more restriction endonucleases, andfragment length sizes are determined by gel electrophoresis andcompared. Differences in fragment length sizes between sample andcontrol DNA indicates mutations in the sample DNA. Moreover, the use ofsequence specific ribozymes (see, for example, U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in PCIP can be identified byhybridizing a sample and control nucleic acids, e.g., DNA or RNA, tohigh density arrays containing hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al. (1996) Human Mutation 7:244-255; Kozal, M.J. et al. (1996) Nature Medicine 2:753-759). For example, geneticmutations in PCIP can be identified in two dimensional arrays containinglight-generated DNA probes as described in Cronin, M. T. et al. supra.Briefly, a first hybridization array of probes can be used to scanthrough long stretches of DNA in a sample and control to identify basechanges between the sequences by making linear arrays of sequentialoverlapping probes. This step allows the identification of pointmutations. This step is followed by a second hybridization array thatallows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the PCIP gene anddetect mutations by comparing the sequence of the sample PCIP with thecorresponding wild-type (control) sequence. Examples of sequencingreactions include those based on techniques developed by Maxam andGilbert ((1977) Proc. Natl. Acad. Sci. USA 74:560) or Sanger ((1977)Proc. Natl. Acad. Sci. USA 74:5463). It is also contemplated that any ofa variety of automated sequencing procedures can be utilized whenperforming the diagnostic assays ((1995) Biotechniques 19:448),including sequencing by mass spectrometry (see, e.g., PCT InternationalPublication No. WO 94/16101; Cohen et al. (1996) Adv. Chromatogr.36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol.38:147-159).

Other methods for detecting mutations in the PCIP gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science230:1242). In general, the art technique of “mismatch cleavage” startsby providing heteroduplexes of formed by hybridizing (labeled) RNA orDNA containing the wild-type PCIP sequence with potentially mutant RNAor DNA obtained from a tissue sample. The double-stranded duplexes aretreated with an agent which cleaves single-stranded regions of theduplex such as which will exist due to basepair mismatches between thecontrol and sample strands. For instance, RNA/DNA duplexes can betreated with RNase and DNA/DNA hybrids treated with S1 nuclease toenzymatically digesting the mismatched regions. In other embodiments,either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine orosmium tetroxide and with piperidine in order to digest mismatchedregions. After digestion of the mismatched regions, the resultingmaterial is then separated by size on denaturing polyacrylamide gels todetermine the site of mutation. See, for example, Cotton et al. (1988)Proc. Natl Acad Sci USA 85:4397; Saleeba et al. (1992) Methods Enzymol217:286-295. In a preferred embodiment, the control DNA or RNA can belabeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in PCIP cDNAs obtained fromsamples of cells. For example, the mutY enzyme of E. coli cleaves A atG/A mismatches and the thymidine DNA glycosylase from HeLa cells cleavesT at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a PCIP sequence,e.g., a wild-type PCIP sequence, is hybridized to a cDNA or other DNAproduct from a test cell(s). The duplex is treated with a DNA mismatchrepair enzyme, and the cleavage products, if any, can be detected fromelectrophoresis protocols or the like. See, for example, U.S. Pat. No.5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in PCIP genes. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton(1993) Mutat. Res. 285:125-144; and Hayashi (1992) Genet. Anal. Tech.Appl. 9:73-79). Single-stranded DNA fragments of sample and control PCIPnucleic acids will be denatured and allowed to renature. The secondarystructure of single-stranded nucleic acids varies according to sequence,the resulting alteration in electrophoretic mobility enables thedetection of even a single base change. The DNA fragments may be labeledor detected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

In yet another embodiment the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner (1987)Biophys Chem 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation isplaced centrally and then hybridized to target DNA under conditionswhich permit hybridization only if a perfect match is found (Saiki etal. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA86:6230). Such allele specific oligonucleotides are hybridized to PCRamplified target DNA or a number of different mutations when theoligonucleotides are attached to the hybridizing membrane and hybridizedwith labeled target DNA.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al. (1992) Mol. Cell Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany (1991) Proc. Natl. Acad. Sci USA 88:189). In suchcases, ligation will occur only if there is a perfect match at the 3′end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a PCIP gene.

Furthermore, any cell type or tissue in which PCIP is expressed may beutilized in the prognostic assays described herein.

3. Monitoring of Effects During Clinical Trials

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a PCIP protein (e.g., the modulation of membraneexcitability or resting potential) can be applied not only in basic drugscreening, but also in clinical trials. For example, the effectivenessof an agent determined by a screening assay as described herein toincrease PCIP gene expression, protein levels, or upregulate PCIPactivity, can be monitored in clinical trials of subjects exhibitingdecreased PCIP gene expression, protein levels, or downregulated PCIPactivity. Alternatively, the effectiveness of an agent determined by ascreening assay to decrease PCIP gene expression, protein levels, ordownregulate PCIP activity, can be monitored in clinical trials ofsubjects exhibiting increased PCIP gene expression, protein levels, orupregulated PCIP activity. In such clinical trials, the expression oractivity of a PCIP gene, and preferably, other genes that have beenimplicated in, for example, a potassium channel associated disorder canbe used as a “read out” or markers of the phenotype of a particularcell.

For example, and not by way of limitation, genes, including PCIP, thatare modulated in cells by treatment with an agent (e.g., compound, drugor small molecule) which modulates PCIP activity (e.g., identified in ascreening assay as described herein) can be identified. Thus, to studythe effect of agents on potassium channel associated disorders, forexample, in a clinical trial, cells can be isolated and RNA prepared andanalyzed for the levels of expression of PCIP and other genes implicatedin the potassium channel associated disorder, respectively. The levelsof gene expression (e.g., a gene expression pattern) can be quantifiedby northern blot analysis or RT-PCR, as described herein, oralternatively by measuring the amount of protein produced, by one of themethods as described herein, or by measuring the levels of activity ofPCIP or other genes. In this way, the gene expression pattern can serveas a marker, indicative of the physiological response of the cells tothe agent. Accordingly, this response state may be determined before,and at various points during treatment of the individual with the agent.

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agent(e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleicacid, small molecule, or other drug candidate identified by thescreening assays described herein) including the steps of (i) obtaininga pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a PCIP protein,mRNA, or genomic DNA in the preadministration sample; (iii) obtainingone or more post-administration samples from the subject; (iv) detectingthe level of expression or activity of the PCIP protein, mRNA, orgenomic DNA in the post-administration samples; (v) comparing the levelof expression or activity of the PCIP protein, mRNA, or genomic DNA inthe pre-administration sample with the PCIP protein, mRNA, or genomicDNA in the post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of PCIP to higher levels than detected, i.e., toincrease the effectiveness of the agent. Alternatively, decreasedadministration of the agent may be desirable to decrease expression oractivity of PCIP to lower levels than detected, i.e. to decrease theeffectiveness of the agent. According to such an embodiment, PCIPexpression or activity may be used as an indicator of the effectivenessof an agent, even in the absence of an observable phenotypic response.

D. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant PCIP expression oractivity. With regards to both prophylactic and therapeutic methods oftreatment, such treatments may be specifically tailored or modified,based on knowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”.) Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the PCIP molecules ofthe present invention or PCIP modulators according to that individual'sdrug response genotype. Pharmacogenomics allows a clinician or physicianto target prophylactic or therapeutic treatments to patients who willmost benefit from the treatment and to avoid treatment of patients whowill experience toxic drug-related side effects.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant PCIPexpression or activity, by administering to the subject a PCIP or anagent which modulates PCIP expression or at least one PCIP activity.Subjects at risk for a disease which is caused or contributed to byaberrant PCIP expression or activity can be identified by, for example,any or a combination of diagnostic or prognostic assays as describedherein. Administration of a prophylactic agent can occur prior to themanifestation of symptoms characteristic of the PCIP aberrancy, suchthat a disease or disorder is prevented or, alternatively, delayed inits progression. Depending on the type of PCIP aberrancy, for example, aPCIP, PCIP agonist or PCIP antagonist agent can be used for treating thesubject. The appropriate agent can be determined based on screeningassays described herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating PCIPexpression or activity for therapeutic purposes. Accordingly, in anexemplary embodiment, the modulatory method of the invention involvescontacting a cell with a PCIP or agent that modulates one or more of theactivities of PCIP protein activity associated with the cell. An agentthat modulates PCIP protein activity can be an agent as describedherein, such as a nucleic acid or a protein, a naturally-occurringtarget molecule of a PCIP protein (e.g., a PCIP substrate), a PCIPantibody, a PCIP agonist or antagonist, a peptidomimetic of a PCIPagonist or antagonist, or other small molecule. In one embodiment, theagent stimulates one or more PCIP activities. Examples of suchstimulatory agents include active PCIP protein and a nucleic acidmolecule encoding PCIP that has been introduced into the cell. Inanother embodiment, the agent inhibits one or more PCIP activities.Examples of such inhibitory agents include antisense PCIP nucleic acidmolecules, anti-PCIP antibodies, and PCIP inhibitors. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant expression or activity of a PCIP protein or nucleic acidmolecule. Examples of such disorders include CNS disorders such asneurodegenerative disorders, e.g., Alzheimer's disease, dementiasrelated to Alzheimer's disease (such as Pick's disease), Parkinson's andother Lewy diffuse body diseases, multiple sclerosis, amyotrophiclateral sclerosis, progressive supranuclear palsy, epilepsy, andJakob-Creutzfieldt disease; psychiatric disorders, e.g., depression,schizophrenic disorders, Korsakoff's psychosis, mania, anxietydisorders, bipolar affective disorders, or phobic disorders; learning ormemory disorders, e.g., amnesia or age-related memory loss; neurologicaldisorders, e.g., migraine; pain disorders, e.g., hyperalgesia or painassociated with muscoloskeletal disorders; spinal cord injury; stroke;and head trauma. In one embodiment, the method involves administering anagent (e.g., an agent identified by a screening assay described herein),or combination of agents that modulates (e.g., upregulates ordownregulates) PCIP expression or activity. In another embodiment, themethod involves administering a PCIP protein or nucleic acid molecule astherapy to compensate for reduced or aberrant PCIP expression oractivity.

A preferred embodiment of the present invention involves a method fortreatment of a PCIP associated disease or disorder which includes thestep of administering a therapeutically effective amount of a PCIPantibody to a subject. As defined herein, a therapeutically effectiveamount of antibody (i.e., an effective dosage) ranges from about 0.001to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight,more preferably about 0.1 to 20 mg/kg body weight, and even morepreferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7mg/kg, or 5 to 6 mg/kg body weight. The skilled artisan will appreciatethat certain factors may influence the dosage required to effectivelytreat a subject, including but not limited to the severity of thedisease or disorder, previous treatments, the general health and/or ageof the subject, and other diseases present. Moreover, treatment of asubject with a therapeutically effective amount of an antibody caninclude a single treatment or, preferably, can include a series oftreatments. In a preferred example, a subject is treated with antibodyin the range of between about 0.1 to 20 mg/kg body weight, one time perweek for between about 1 to 10 weeks, preferably between 2 to 8 weeks,more preferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. It will also be appreciated that the effectivedosage of antibody used for treatment may increase or decrease over thecourse of a particular treatment. Changes in dosage may result from theresults of diagnostic assays as described herein.

Stimulation of PCIP activity is desirable in situations in which PCIP isabnormally downregulated and/or in which increased PCIP activity islikely to have a beneficial effect. For example, stimulation of PCIPactivity is desirable in situations in which a PCIP is downregulatedand/or in which increased PCIP activity is likely to have a beneficialeffect. Likewise, inhibition of PCIP activity is desirable in situationsin which PCIP is abnormally upregulated and/or in which decreased PCIPactivity is likely to have a beneficial effect.

3. Pharmacogenomics

The PCIP molecules of the present invention, as well as agents, ormodulators which have a stimulatory or inhibitory effect on PCIPactivity (e.g., PCIP gene expression) as identified by a screening assaydescribed herein can be administered to individuals to treat(prophylactically or therapeutically) potassium channel associateddisorders associated with aberrant PCIP activity (e.g, CNS disorderssuch as neurodegenerative disorders, e.g., Alzheimer's disease,dementias related to Alzheimer's disease (such as Pick's disease),Parkinson's and other Lewy diffuse body diseases, multiple sclerosis,amyotrophic lateral sclerosis, progressive supranuclear palsy, epilepsy,and Jakob-Creutzfieldt disease; psychiatric disorders, e.g., depression,schizophrenic disorders, Korsakoff's psychosis, mania, anxietydisorders, bipolar affective disorders, or phobic disorders; learning ormemory disorders, e.g., amnesia or age-related memory loss; neurologicaldisorders, e.g., migraine; pain disorders, e.g., hyperalgesia or painassociated with muscoloskeletal disorders; spinal cord injury; stroke;and head trauma). In conjunction with such treatment, pharmacogenomics(i.e., the study of the relationship between an individual's genotypeand that individual's response to a foreign compound or drug) may beconsidered. Differences in metabolism of therapeutics can lead to severetoxicity or therapeutic failure by altering the relation between doseand blood concentration of the pharmacologically active drug. Thus, aphysician or clinician may consider applying knowledge obtained inrelevant pharmacogenomics studies in determining whether to administer aPCIP molecule or PCIP modulator as well as tailoring the dosage and/ortherapeutic regimen of treatment with a PCIP molecule or PCIP modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drugs target is known (e.g., a PCIPprotein of the present invention), all common variants of that gene canbe fairly easily identified in the population and it can be determinedif having one version of the gene versus another is associated with aparticular drug response.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a PCIP molecule orPCIP modulator of the present invention) can give an indication whethergene pathways related to toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a PCIP molecule orPCIP modulator, such as a modulator identified by one of the exemplaryscreening assays described herein.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures and the Sequence Listing areincorporated herein by reference.

EXAMPLES

The following materials and methods were used in the Examples.

Strains, Plasmids, Bait cDNAs, and General Microbiological Techniques

Basic yeast strains (HF7c, Y187,) bait (pGBT9) and fish (pACT2) plasmidsused in this work were purchased from Clontech (Palo Alto, Calif.).cDNAs encoding rat Kv4.3, Kv4.2, and Kv1.1, were provided byWyeth-Ayerst Research (865 Ridge Rd., Monmouth Junction, N.J. 08852)Standard yeast media including synthetic complete medium lackingL-leucine, L-tryptophan, and L-histidine were prepared and yeast geneticmanipulations were performed as described (Sherman (1991) Meth. Enzymol.194:3-21). Yeast transformations were performed using standard protocols(Gietz et al. (1992) Nucleic Acids Res. 20:1425; Ito et al (1983) J.Bacteriol. 153:163-168). Plasmid DNAs were isolated from yeast strainsby a standard method (Hoffman and Winston (1987) Gene 57:267-272).

Bait and Yeast Strain Construction

The first 180 amino acids of rKv4.3 (described in Serdio P. et al.(1996) J. Neurophys 75:2174-2179) were amplified by PCR and cloned inframe into pGBT9 resulting in plasmid pFWA2, (hereinafter “bait”). Thisbait was transformed into the two-hybrid screening strain HF7c andtested for expression and self-activation. The bait was validated forexpression by Western blotting. The rKv4.3 bait did not self-activate inthe presence of 10 mM 3-amino-1,2,3-Triazole (3-AT).

Library Construction

Rat mid brain tissue was provided by Wyeth-Ayerst Research (MonmouthJunction, N.J.). Total cellular RNA was extracted from the tissues usingstandard techniques (Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., (1989)). mRNA was prepared using a Poly-A Spin mRNA Isolation Kitfrom New England Biolabs (Beverly, Mass.). cDNA from the mRNA sample wassynthesized using a cDNA Synthesis Kit from Stratagene (La Jolla,Calif.) and ligated into pACT2's EcoRI and XhoI sites, giving rise to atwo-hybrid library.

Two-Hybrid Screening

Two-hybrid screens were carried out essentially as described in Bartel,P. et al. (1993) “Using the Two-Hybrid System to DetectPolypeptide-Polypeptide Interactions” in Cellular Interactions inDevelopment: A Practical Approach, Hartley, D. A. ed. Oxford UniversityPress, Oxford, pp. 153-179, with a bait-library pair of rkv4.3 bait-ratmid brain library. A filter disk beta-galactosidase (beta-gal) assay wasperformed essentially as previously described (Brill et al. (1994) Mol.Biol. Cell. 5:297-312). Clones that were positive for both reporter geneactivity (His and beta-galactosidase) were scored and fish, plasmidswere isolated from yeast, transformed into E. coli strain KC8, DNAplasmids were purified and the resulting plasmids were sequenced byconventional methods (Sanger F. et al. (1977) PNAS, 74: 5463-67).

Specificity Test

Positive interactor clones were subjected to a binding specificity testwhere they were exposed to a panel of related and unrelated baits by amating scheme previously described (Finley R. L. Jr. et al. (1994) PNAS,91(26):12980-12984). Briefly, positive fish plasmids were transformedinto Y187 and the panel of baits were transformed into HF7c. Transformedfish and bait cells were streaked out as stripes on selective mediumplates, mated on YPAD plates, and tested for reporter gene activity.

Analysis

PCIP nuleotides were analyzed for nucleic acid hits by the BLASTN1.4.8MP program (Altschul et al. (1990) Basic Local Alignment SearchTool. J. Mol. Biol. 215:403-410). PCIP proteins were analyzed forpolypeptide hits by the BLASTP 1.4.9MP program.

Example 1

Identification of Rat PCIP cDNAs

The Kv4.3 gene coding sequence (coding for the first 180 amino acids)was amplified by PCR and cloned into pGBT9 creating a GAL4 DNA-bindingdomain-Kv4.3(1-180) gene fusion (plasmid pFWA2). HF7c was transformedwith this construct. The resulting strain grew on synthetic completemedium lacking L-tryptophan but not on synthetic complete medium lackingL-tryptophan and L-histidine in the presence of 10 mM 3-AT demonstratingthat the {GAL4 DNA-binding domain}-{vKv4.3(1-180)} gene fusion does nothave intrinsic transcriptional activation activity higher than thethreshhold allowed by 10 mM 3-AT.

In this example, a yeast two-hybrid assay was performed in which aplasmid containing a {GAL4 DNA-binding domain}-{rKv4.3(1-180)} genefusion was introduced into the yeast two-hybrid screening strain HF7cdescribed above. HF7c was then transformed with the rat mid braintwo-hybrid library. Approximately six million transformants wereobtained and plated in selection medium. Colonies that grew in theselection medium and expressed the beta-galactosidase reporter gene werefarther characterized and subjected to retransformation and specificityassays. The retransformation and specificity tests yielded three PCIPclones (rat 1v, 8t, and 9qm) that were able to bind to the Kv4.3polypeptide.

The full length sequences for the rat 1v gene, and partial sequences for8t and 9q genes were derived as follows. The partial rat PCIP sequenceswere used to prepare probes, which were then used to screen, forexample, rat mid brain cDNA libraries. Positive clones were identified,amplified and sequenced using standard techniques, to obtain the fulllength sequence. Additionally, a rapid amplification of the existing ratPCIP cDNA ends (using for example, 5′ RACE, by Gibco, BRL) was used tocomplete the 5′ end of the transcript.

Example 2

Identification of Human 1v cDNA

To obtain the human 1v nucleic acid molecule, a cDNA library made from ahuman hippocampus (Clontech, Palo Alto, Calif.) was screened under lowstringency conditions as follows: Prehybridization for 4 hours at 42° C.in Clontech Express Hyb solution, followed by overnight hybridization at42° C. The probe used was a PCR-generated fragment including nucletides49-711 of the rat sequence labeled with ³²P dCTP. The filters werewashed 6 times in 2×SSC/0.1% SDS at 55° C. The same conditions were usedfor secondary screening of the positive isolates. Clones thus obtainedwere sequenced using an ABI automated DNA Sequencing system, andcompared to the rat sequences shown in SEQ ID NO:3 as well as to knownsequences from the GenBank database. The largest clone from the libraryscreen was subsequently subcloned into pBS-KS+ (Stratagene, La Jolla,Calif.) for sequence verification. The 515 base pair clone wasdetermined to represent the human homolog of the 1v gene, encompasing211 base pairs of 5′ UTR and a 304 base pair coding region. To generatethe full-length cDNA, 3′ RACE was used according to the manufacturersinstructions (Clontech Advantage PCR kit).

Example 3

Isolation and Characterization of 1v Splice Variants

The mouse 1v shown in SEQ ID NO:5 and the rat 1vl splice variant shownin SEQ ID NO:7 was isolated using a two-hybrid assay as described inExample 1. The mouse 1vl splice variant shown in SEQ ID NO:7 wasisolated by screening a mouse brain cDNA library, and the rat 1vn splicevariant shown in SEQ ID NO:11 was isolated by BLAST searching.

Example 4

Isolation and Identification of 9q and Other PCIPs

Rat 9ql (SEQ ID NO:15) was isolated by database mining, rat 9qm (SEQ IDNO:21) was isolated by a two-hybrid assay, and rat 9qc (SEQ ID NO:27)was identified by database mining. Human 9ql (SEQ ID NO:13), and human9qs (SEQ ID NO:23) were identified as described in Example 2. Mouse 9ql(SEQ ID NO:17), monkey 9qs (SEQ ID NO:25), human p193 (SEQ ID NO:39),rat p19 (SEQ ID NO:33), and mouse p19 (SEQ ID NO:35) were identified bydatabase mining. Rat 8t (SEQ ID NO:29) was identified using a two-hybridassay. The sequence of W28559 (SEQ ID NO:37) was identified by databasemining and sequencing of the identified EST with Genbank AccessionNumber AI352454. The protein sequence was found to contain a 41 aminoacid region with strong homology to 1v, 9ql, and p19 (see alignment inFIG. 25). However, downstream of this homologous region the sequencediverges from that of the PCIP family. This sequence could represent agene which possesses a 41 amino acid domain with homology to a similardomain found in the PCIP family members.

The human genomic 9q sequence (SEQ ID NOs:46 and 47) was isolated byscreening a BAC genomic DNA library (Reasearch Genetics) using primerswhich were designed based on the sequence of the human 9qm cDNA. Twopositive clones were identified (448O2 and 721I17) and sequenced.

Example 5

Expression of 1v, 8t, and 9q mRNA in Rat Tissues

Rat and mouse multiple tissue Northern blots (Clontech) were probed witha [³²P]-labeled cDNA probe directed at the 5′-untranslated and 5′-codingregion of the rat 1v sequence (nucleotides 35-124; SEQ ID NO:3) (thisprobe is specific for rat 1v and rat 1vl), the 5′ coding region of the8t sequence (nucleotides 1-88; SEQ ID NO:29) (this probe is specific for8t), or the 5′ end of the rat 9qm sequence (nucleotides 1-195; SEQ IDNO:21) (this probe is specific for all 9q isoforms, besides 8t). Blotswere hybridize using standard techniques. Northern blots hybridized withthe rat 1v probe revealed a single band at 2.3 kb only in the lanecontaining brain RNA, suggesting that 1v expression is brain specific.Northern blots probed with the rat 8t probe revealed a major band at 2.4kb. The rat 8t band was most intense in the lane containing heart RNAand there was also a weaker band in the lane containing brain RNA.Northern blots hybridized with the 9q cDNA probe revealed a major bandat 2.5 kb and a minor band at over 4 kb with predominant expression inbrain and heart. The minor band may represent incompletely spliced orprocessed 9q mRNA. The results from the northern blots further indicatedthat p19 is expressed predominantly in the heart.

Example 6

Expression of 1v, 8t, and 9q in Brain

Expression of the rat 1v and 8t/9q genes in the brain was examined by insitu hybridization histochemistry (ISHH) using [³⁵S]-labeled cRNA probesand a hybridization procedure identical to that described in Rhodes etal. (1996) J. Neurosci., 16:4846-4860. Templates for preparing the cRNAprobes were generated by standard PCR methods. Briefly, oligonucleotideprimers were designed to amplify a fragment of 3′- or 5′-untranslatedregion of the target cDNA and in addition, add the promoter recognitionsequences for T7 and T3 polymerase. Thus, to generate a 300 nucleotideprobe directed at the 3′-untranslated region of the 1v mRNA, we used thefollowing primers:

5-TAATACGACTCACTATAGGGACTGGCCATCCTGCTCTCAG-3  (T7, forward, sense; SEQID NO:42)

5-ATTAACCCTCACTAAAGGGACACTACTGTTTAAGCTCAAG-3  (T3, reverse, antisense;SEQ ID NO:43).

The underlined bases correspond to the T7 and T3 promoter sequences. Togenerate a probe directed at a 325 bp region of 3′-untranslated sequenceshared by the 8t and 9q mRNAs, the following primers were used:

5-TAATACGACTCACTATAGGGCACCTCCCCTCCGGCTGTTC-3  (T7, forward, sense; SEQID NO:44)

5-ATTAACCCTCACTAAAGGGAGAGCAGCAGCATGGCAGGGT-3  (T3, reverse, antisense;SEQ ID NO:45).

Autoradiograms of rat brain tissue sections processed for ISHHlocalization of 1v or 8t/9q mRNA expression revealed that 1v mRNA isexpressed widely in brain in a pattern consistent with labeling ofneurons as opposed to glial or endothelial cells. 1v mRNA is highlyexpressed in cortical, hippocampal, and striatal interneurons, thereticlar nucleus of the thalamus, the medial habenula, and in cerebellargranule cells. 1v mRNA is expressed at moderate levels in midbrainnuclei including the substantia nigra and superior colliculus, inseveral other thalamic nuclei, and in the medial septal and diagonalband nuclei of the basal forebrain.

Because the probe used to analyze the expression of 8t and 9q hybridizesto a region of the 3-untranslated region that is identical in the 8t and9q mRNAs, this probe generates a composite image that reveals that 8t/9qmRNA is expressed widely in brain in a pattern that partly overlaps withthat for 1v as described above. However, 8t/9q mRNA is highly expressedin the striatum, hippocampal formation, cerebellar granule cells, andneocortex. 8t/9q mRNA is expressed at moderate levels in the midbrain,thalamus, and brainstem. In may of these areas, 8t./9q mRNA appears tobe concentrated in interneurons in addition to principal cells, and inall regions 8t/9q expression appears to be concentrated in neurons asapposed to glial cells.

Single- and double-label immunohistochemistry revealed that the PCIP andKv4 polypeptides are precisely colocalized in many of the cell types andbrain regions where PCIP and Kv4 mRNAs are coexpressed. For example, 9qmcolocalized with Kv4.2 in the somata and dendrites of hippocampalgranule and pyramidal cells, neurons in the medial habenular nucleus andin cerebellar basket cells, while 1v colocalized with Kv4.3 in layer IIneurons of posterior cingulate cortex, hippocampal interneurons, and ina subset of cerebellar granule cells. Immunoprecipitation analysesindicated that 1v and 9qm are coassociated with Kv4 α-subunits in ratbrain membranes.

Example 7

Co-Association of PCIPs and Kv4 Channels in COS and CHO Cells

COS1 and CHO cells were transiently transfected with individual PCIPs(KChIP1, KChIP2, KChIP3) alone or together with Kv4.2 or Kv4.3 using thelipofectamine plus procedure essentially as described by themanufacturer (Boehringer Mannheim). Forty-eight hours after thetransfection, cells were washed, fixed, and processed forimmunofluorescent visualization as described previously (Bekele-Arcuriet al. (1996) Neuropharmacology, 35:851-865). Affinity-purified rabbitpolyclonal or mouse monoclonal antibodies to the Kv4 channel or the PCIPprotein were used for immunofluorescent detection of the targetproteins.

When expressed alone, the PCIPs were diffusely distributed throughoutthe cytoplasm of COS-1 and CHO cells, as would be expected forcytoplasmic proteins. In contrast, when expressed alone, the Kv4.2 andKv4.3 polypeptides were concentrated within the perinuclear ER and Golgicompartments, with some immunoreactivity concentrated in the outermargins of the cell. When the PCIPs were coexpressed with Kv4α-subunits, the characteristic diffuse PCIP distribution changeddramatically, such that the PCIPs precisely colocalized with the Kv4α-subunits. This redistribution of the PCIPs did not occur when theywere coexpressed with the Kv1.4 α-subunit, indicating that altered PCIPlocalization is not a consequence of overexpression and that these PCIPsassociate specifically with Kv4-family α-subunits.

To verify that the PCIP and Kv4 polypeptides are tightly associated andnot simply colocalized in co-transfected cells, reciprocalimmunoprecipitation analyses were performed using the PCIP andchannel-specific antibodies described above. All three PCIP polypeptidescoassociated with Kv4 α-subunits in cotransfected cells, as evidenced bythe ability of anti-Kv4.2 and anti-Kv4.3 antibodies to immunoprecipitatethe KChIP1, KChIP2, and KChIP3 proteins from lysates prepared fromcotransfected cells, and by the ability of anti-PCIP antibodies toimmunoprecipitate Kv4.2 and Kv4.3 α-subunits from these same lysates.The cells were lysed in buffer containing detergent and proteaseinhibitors, and prepared for immunoprecipitation reactions essentiallyas described previously (Nakahira et al. (1996) J. Biol. Chem.,271:7084-7089). Immunoprecipitations were performed as described inNakahira et al. (1996) J. Biol. Chem., 271:7084-7089 and in Harlow E.and Lane, D., Antibodies:A Laboratory Manual, Cold Spring HarborLaboratory, c1988. The products resulting from the immunoprecipitationwere size fractionated by SDS-PAGE and transferred to nitrocellulosefilters using standard procedures.

To confirm that the cytoplasmic N-terminus of Kv4 channels is sufficientfor the interaction with the PCIPs KChIP1 or KChIP2 were co-expressedwith a Kv4.3 mutant (Kv4.3ΔC) that lacks the entire 219 amino acidcytoplasmic C-terminal tail. In transiently transfected COS-1 cells, theKv4.3ΔC mutant was extensively trapped within the perinuclear ER andGolgi: little or no staining was observed at the outer margins of thecell. Nonetheless, KChIP1 and KChIP2 precisely colocalized with Kv4.3ΔCin cotransfected cells, and moreover, Kv4.3ΔC was efficientlycoinmunoprecipitated by PCIP antibodies, indicating that the interactionof these PCIPs with Kv4 α-subunits does not require the cytoplasmicC-terminus of the channel.

Example 8

Co-Association of PCIPs and Kv4 Channels in Native Tissues

To determine whether PCIPs colocalize and co-associate with Kv4 subunitsin native tissues, Kv4- and PCIP-specific antibodies were used forsingle and double-label immunohistochemical analyses and for reciprocalcoimmunoprecipitation analyses of rat brain membranes.Immunohistochemical staining of rat brain sections indicated that KChIP1and KChIP2 colocalize with Kv4.2 and Kv4.3 in a region and celltype-specific manner. For example, KChIP1 colocalized with Kv4.3 inhippocampal interneurons, cerebellar granule cells, and cerebellarglomeruli, a specialized synaptic arrangement between the dendrites ofcerebellar basket and golgi cells and mossy fiber terminals. KChIP2colocalized with Kv4.3 and Kv4.2 in the dendrites of granule cells inthe dentate gyrus, in the apical and basal dendrites of hippocampal andneocortical pyramidal cells, and in several subcortical structuresincluding the striatum and superior colliculus. Co-immunoprecipitationanalyses performed using synaptic membranes prepared from whole ratbrain revealed that the PCIPs (KChIPs 1, 2, and 3) are tightlyassociated with Kv4.2 and Kv4.3 in brain K+ channel complexes. Anti-PCIPantibodies immunoprecipitated Kv4.2 and Kv4.3 from brain membranes, andanti-Kv4.2 and Kv4.3 antibodies immunoprecipitated the PCIPs. None ofthe PCIP polypeptides were immunoprecipitated by anti-Kv2.1 antibodies,indicating that the association of these PCIPs with brain Kv channelsmay be specific for Kv4 α-subunits. Taken together, these anatomical andbiochemical analyses indicate that these PCIPs are integral componentsof native Kv4 channel complexes.

Example 9

PCIPs are Cacium Binding Proteins

To determine whether KChIPs 1, 2, and 3 bind Ca2+, GST-fusion proteinswere generated for each PCIP and the ability of the GST-PCIP proteins,as well as the recombinant PCIP polypeptides enzymatically cleaved fromGST, to bind ⁴⁵Ca2+ was examined using a filter overlay assay (describedin, for example, Kobayashi et al. (1993) Biochem. Biophys. Res. Commun.189(1):511-7). All three PCIP polypeptides, but not an unrelatedGST-fusion protein, display strong ⁴⁵Ca2+ binding in this assay.Moreover, all three PCIP polypeptides display a Ca2+-dependent mobilityshift on SDS-PAGE, indicating that like the other members of thisfamily, KChIPs 1, 2 and 3 are in fact Ca2+-binding proteins (Kobuyashiet al. (1993) supra; Buxbaum et al. Nef (1996). Neuron-specific calciumsensors (the NCS-1 subfamily). In: Celio MR (ed) Guidebook to thecalcium-binding proteins. Oxford University Press, New York, pp94-98;Buxbaum J. D., et al. (1998) Nature Med. 4(10):1177-81.

Example 10

Electrophysiological Chatacterization of PCIPs

Because PCIPs, e.g., KChIP1 (1v), KChIP2 (9ql), and KChIP3 (p19),colocalize and coassociate with Kv4 α-subunits in brain, anothercritical question was to determine whether these PCIPs alter theconductance properties of Kv4 channels. To address this issue, Kv4.2 andKv4.3 were expressed alone and in combination with individual PCIPs. CHOcells were transiently-transfected with cDNA using the DOTAP lipofectionmethod as described by the manufacturer (Boehringer Mannheim, Inc.).Transfected cells were identified by cotransfecting enhanced GFP alongwith the genes of interest and subsequently determining if the cellscontained green GFP fluorescence. Currents in CHO cells were measuredusing the patch-clamp technique (Hamill et al. 1981. Pfluegers Arch.391:85-100).

Transient transfection of the rat Kv4.2 α-subunit in CHO cells resultedin expression of a typical A-type K+ conductance. Coexpression of Kv4.2with KChIP1 revealed several dramatic effects of KChIP1 on the channel(FIG. 41 and Table 1). First, the amplitude of the Kv4.2 currentincreased approximately 7.5 fold in the presence of KChIP1 (amplitude ofKv4.2 alone=0.60±0.096 nA/cell; Kv4.2+KChIP1=4.5±0.55 nA/cell). Whenconverted into current density by correcting for cell capacitance, ameasure of cell surface membrane area, the Kv4.2 current densityincreased 12 fold with coexpression of KChIP1 (Kv4.2 alone=25.5±3.2pA/pF; Kv4.2+KChIP1=306.9±57.9 pA/pF), indicating that KChIPs promoteand/or stabilize Kv4.2 surface expression. Together with this increasein current density, a dramatic leftward shift in the threshold foractivation of Kv4.2 currents was observed in cells expressing Kv4.2 andKChIP1 (activation V1/2 for Kv4.2 alone=20.8±7.0 mV,Kv4.2+KChIP1=−12.1±1.4 mV). Finally, the kinetics of Kv4.2 inactivationslowed considerably when Kv4.2 was coexpressed with KCHIP1 (inactivationtime constant of Kv4.2 alone=28.2±2.6 ms; Kv4.2+KChIP1=104.1±10.4 ms),while channels recovered from inactivation much more rapidly in cellsexpressing both Kv4.2 and KChIP1 (recovery tau=53.6±7.6 ms) versus cellsexpressing Kv4.2 alone (recovery tau=272.2±26.1 ms).

KChIPs1, 2 and 3 have distinct N-termini but share considerable aminoacid identity within the C-terminal “core” domain. Despite theirdistinct N-termini, the effects of KChIP2 and KChIP3 on Kv4.2 currentdensity and kinetics were strikingly similar to those produced by KChIP1(Table1). Thus to confirm that the conserved C-terminal core domain,which contains all three EF-hands, is sufficient to modulate Kv4 currentdensity and kinetics, N-terminal truncation mutants of KChIP1 and KChIP2were prepared. The KChIP1ΔN2-31 and KChIP2ΔN2-67 mutants truncatedKChIP1 and KChIP2, respectively, to the C-terminal 185 amino acid coresequence. Coexpression of KChIP1ΔN2-31 or KChIP2ΔN2-67 with Kv4.2 in CHOcells produced changes in Kv4.2 current density and kinetics that wereindistinguishable from the effects produced by full-length KChIP1 orKChIP2 (Table1).

To investigate whether the modulatory effects of these KChIPs arespecific for Kv4 channels, KChIP1 was coexpressed with Kv1.4 and Kv2.1in Xenopus oocytes. Xenopus oocytes were injected with 1-3 ng/oocyte ofcRNA which was prepared using standard in vitro transcription techniques(Sambrook et al. 1989. Molecular Cloning: a laboratory manual, ColdSpring Harbor Press). Currents in oocytes were measured with atwo-electrode voltage clamp. KChIP1 did not appear to have any effect onKv1. 4 or Kv2.1 currents (Table2), indicating that these functionaleffects may be specific for Kv4 channels. As a final control for theKCHIP effects and to verify that the KChIPs' effects on Kv4 currents areindependent of expression system, the above kinetic analyses wererepeated after expressing Kv4.3 and KChIP mRNAs in Xenopus oocytes. Theeffects KChIP1 on for Kv4.3 in the oocyte system were strikingly similarto those on Kv4.2 in CHO cells (Table1).

Since these KChIPs bind Ca2+, another important question is to determinewhether the effects of KChIP1 on Kv4.2 currents are Ca2+-dependent. Thisquestion was addressed indirectly by introducing point mutations withineach of KChIP1's EF-hand domains: one mutant has point mutations in thefirst two EF hands (D₁₉₉ to A, G₁₀₄ to A, D₁₃₅ to A, and G₁₄₀ to A) andthe other one has point muations in all three EF hands (D₁₉₉ to A, G₁₀₄to A, D₁₃₅ to A, G₁₄₀ to A, D₁₈₃ to A, and G₁₈₈ to A). These mutationssubstituted alanine for the two most highly conserved amino acids withinthe EF-hand consensus (FIG. 25; Linse, S. and Forsen, S. (1995)Determinants that govern high-affinity Calcium binding. In Means, S.(Ed.)Advances in second messenger and phosphoprotein research. New York,Ravens Press,. 30:89-150). Coexpression of this KChIP1 triple EF-handmutant with Kv4.2 or Kv4.3 in COS cells indicated that this mutantcolocalizes and is efficiently coimmunoprecipitated with Kv4 α-subunitsin COS-1 cells. However, these EF-hand point mutations completelyeliminated the effects of KChIP1 on Kv4.2 kinetics (Table1). Takentogether, these results indicate that the binding interaction betweenKChIP1 and Kv4.2 is Ca2+ independent, while modulation of Kv4.2 kineticsby KChIP1 is either Ca2+-dependent or sensitive to structural changesinduced by point mutations within the EF-hand domains.

TABLE 1 Functional effect of KchIPs on Kv4 channels rKv4.2 + rKv4.2 +rKv4.2 + rKv4.2 + KchIP1 rKv4.2 + KchIP2 rKv4.2 + rKv4.3 + CurrentParameter vector KchIP1 ΔN2-31 KchIP2 ΔN2-67 KchIP3 rKv4.3 KchIP1 PeakCurrent  0.60*  4.5*    6.0*  3.3*  5.8*  3.5*  7.7 μA  18.1 μA*(nA/cell at 50 MV)  ±0.096  ±0.055  ±1.1  ±0.45  ±1.1  ±0.99  ±2.6  ±3.8Peak Current Density  25.5 306.9*   407.2* 196.6* 202.6* 161.7* — —(pA/pF at 50 mV)  ±3.2 ±57.9 ±104.8  ±26.6 ±27.5 ±21.8 Inactivation time 28.2 104.1   129.2  95.1* 109.5*  67.2*  56.3 135.0 constant (ms, at 50mV)  ±2.6 ±10.4  ±14.2  ±8.3  ±9.6 ±14.1  ±6.6 ±15.1 Recovery from 272.2 53.6*    98.1*  49.5*  36.1* 126.1* 327.0  34.5* Inactivation Timeconstant *Significantly different from control.

TABLE 2 Functional effects of KChIPs on other Kv channels OocytesOocytes Current Parameter HKv1.4 hKv1.4 + 1v HKv2.1 HKv2.1 + 1v Peak 8.36.5 3.7 2.9 Current (μA/cell ±2.0 ±0.64 ±0.48 ±0.37 at 50 MV)Inactivation 53.2 58.2 1.9 s 1.7 s time constant (ms, at 50 mV) ±2.8±6.6 ±0.079 0.078 Recovery from 1.9 1.6 7.6 7.7 Inactivation timeconstant (sec, at −80 mV) Activation V_(1/2) −21.0 −20.9 12.0 12.4 (mV)Steady-state −48.1 −47.5 −25.3 −23.9 Inactivation V1/2 (mV)

Example 11

Effects of KChIP1 on Surface Expression of KV4-α Subunits in COS-1 Cells

To examine the ability of KCHIP1 to enhance the surface expression ofKv4 channels, the ability of KChIP1 to promote the formation of surfaceco-clusters of Kv4 channels and PSD-95 was monitored. PSD-95 is used tofacilitate the visualization of the complex.

To facilitate the interaction between Kv4.3 and PSD-95, a chimeric Kv4.3subunit (Kv4.3ch) was generated in which the C-terminal 10 amino acidsfrom rKv1.4 (SNAKAVETDV, SEQ ID NO:73) were appended to the C-terminusof Kv4.3. The C-terminal 10 amino acids from rKv1.4 were used becausethey associate with PSD-95 and confer the ability to associate withPSD-95 to the Kv4.3 protein when fused to the Kv4.3 C-terminus.Expression of Kv4.3ch in COS-1 cells revealed that the Kv4.3chpolypeptide was trapped in the perinuclear cytoplasm, with minimaldetectable Kv4.3ch immunoreactivity at the outer margins of the cell.When Kv4.3ch was co-expressed with PSD-95, PSD-95 became trapped in theperinuclear cytoplasm and co-localized with Kv4.3ch. However, whenKChIP1 was co-expressed with Kv4.3ch and PSD-95, large plaque-likesurface co-clusters of Kv4.3ch, KChIP1 and PSD-95 were observed.Triple-label immunofluorescence confirmed that these surface clusterscontain all three polypeptides, and reciprocal co-immunoprecipitationanalyses indicated that the three polypeptides are co-associated inthese surface clusters. Control experiments indicated that KChIP1 doesnot interact with PSD-95 alone, and does not co-localize with Kv1.4 andPSD-95 in surface clusters. Taken together, these data indicate thatKChIP1 may promote the transit of the Kv4.3 subunits to the cellsurface.

Example 12

Characterization of the PCIP Proteins

In this example, the amino acid sequences of the PCIP proteins werecompared to amino acid sequences of known proteins and various motifswere identified.

The 1v polypeptide, the amino acid sequence of which is shown in SEQ IDNO:3 is a novel polypeptide which includes 216 amino acid residues.Domains that are putatively involved in calcium binding (Linse, S. andForsen, S. (1995) Advances in Second Messenger and PhosphoproteinResearch 30, Chapter 3, p89-151, edited by Means, AR., Raven Press,Ltd., New York), were identified by sequence alignment (see FIG. 21).

The 8t polypeptide, the amino acid sequence of which is shown in SEQ IDNO:30 is a novel polypeptide which includes 225 amino acid residues.Calcium binding domains that are putatively involved in calcium binding(Linse, S. and Forsen, S. (1995) Advances in Second Messenger andPhosphoprotein Research 30, Chapter 3, p89-151, edited by Means, AR.,Raven Press, Ltd.; New York), were identified by sequence alignment (seeFIG. 21).

The 9q polypeptide is a novel polypeptide which includes calcium bindingdomains that are putatively involved in calcium binding (Linse, S. andForsen, S. (1995) Advances in Second Messenger and PhosphoproteinResearch 30, Chapter 3, p89-151, edited by Means, AR., Raven Press,Ltd., New York (see FIG. 21).

The p19 polypeptide is a novel polypeptide which includes calciumbinding domains that are putatively involved in calcium binding (Linse,S. and Forsen, S. (1995) Advances in Second Messenger and PhosphoproteinResearch 30, Chapter 3, p89-151, edited by Means, AR., Raven Press,Ltd., New York (see FIG. 21).

A BLASTN 2.0.7 search (Altschul et al. (1990) J. Mol. Biol. 215:403) ofthe nucleotide sequence of rat 1vl revealed that the rat 1vl is similarto the rat cDNA clone RMUAH89 (Accession Number AA849706). The rat 1 vlnucleic acid molecule is 98% identical to the rat cDNA clone RMUAH89(Accession Number AA849706) over nucleotides 1063 to 1488.

A BLASTN 2.0.7 search (Altschul et al. (1990) J. Mol. Biol. 215:403) ofthe nucleotide sequence of human 9ql revealed that the human 9ql issimilar to the human cDNA clone 1309405 (Accession Number AA757119). Thehuman 9 ql nucleic acid molecule is 98% identical to the human cDNAclone 1309405 (Accession Number AA757119) over nucleotides 937 to 1405.

A BLASTN 2.0.7 search (Altschul et al. (1990) .J Mol. Biol. 215:403) ofthe nucleotide sequence of mouse P19 revealed that the mouse P19 issimilar to the Mus musculus cDNA clone MNCb-7005 (Accession NumberAU035979). The mouse P19 nucleic acid molecule is 98% identical to theMus musculus cDNA clone MNCb-7005 (Accession Number AU035979) overnucleotides 1 to 583.

Example 13

Expression of Recombinant PCIP Proteins in Bacterial Cells

In this example, PCIP is expressed as a recombinantglutathione-S-transferase (GST) fusion polypeptide in E. coli and thefusion polypeptide is isolated and characterized. Specifically, PCIP isfused to GST and this fusion polypeptide is expressed in E. coli, e.g.,strain BI21. Expression of the GST-PCIP fusion protein in BI21 isinduced with IPTG. The recombinant fusion polypeptide is purified fromcrude bacterial lysates of the induced BI21 strain by affinitychromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Rat 1v and 9ql were cloned into pGEX-6p-2 (Pharmacia). The resultingrecombinant fusion proteins were expressed in E. coli cells and purifiedfollowing art known methods (described in, for example, CurrentProtocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons:1992). The identities of the purified proteins were verified by westernblot analysis using antibodies raised against peptide epitopes of rat 1vand 9ql.

Example 14

Expression of Recombinant PCIP Proteins in COS Cells

To express the PCIP gene in COS cells, the pcDNA/Amp vector byInvitrogen Corporation (San Diego, Calif.) is used. This vector containsan SV40 origin of replication, an ampicillin resistance gene, an E. colireplication origin, a CMV promoter followed by a polylinker region, andan SV40 intron and polyadenylation site. A DNA fragment encoding theentire PCIP protein and an HA tag (Wilson et al. (1984) Cell 37:767) ora FLAG tag fused in-frame to its 3′ end of the fragment is cloned intothe polylinker region of the vector, thereby placing the expression ofthe recombinant protein under the control of the CMV promoter.

To construct the plasmid, the PCIP DNA sequence is amplified by PCRusing two primers. The 5′ primer contains the restriction site ofinterest followed by approximately twenty nucleotides of the PCIP codingsequence starting from the initiation codon; the 3′ end sequencecontains complementary sequences to the other restriction site ofinterest, a translation stop codon, the HA tag or FLAG tag and the last20 nucleotides of the PCIP coding sequence. The PCR amplified fragmentand the pCDNA/Amp vector are digested with the appropriate restrictionenzymes and the vector is dephosphorylated using the CIAP enzyme (NewEngland Biolabs, Beverly, Mass.). Preferably the two restriction siteschosen are different so that the PCIP gene is inserted in the correctorientation. The ligation mixture is transformed into E. coli cells(strains HB101, DH5a, SURE, available from Stratagene Cloning Systems,La Jolla, Calif., can be used), the transformed culture is plated onampicillin media plates, and resistant colonies are selected. PlasmidDNA is isolated from transformants and examined by restriction analysisfor the presence of the correct fragment.

COS cells are subsequently transfected with the PCIP-pcDNA/Amp plasmidDNA using the calcium phosphate or calcium chloride co-precipitationmethods, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Other suitable methods for transfecting host cells canbe found in Sambrook, J., Fritsh, E. F., and Maniatis, T. MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989. Theexpression of the PCIP polypeptide is detected by radiolabelling(³⁵S-methionine or ³⁵S-cysteine available from NEN, Boston, Mass., canbe used) and immunoprecipitation (Harlow, E. and Lane, D. Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1988) using an HA specific monoclonal antibody. Briefly,the cells are labelled for 8 hours with ³⁵S-methionine (or³⁵S-cysteine). The culture media are then collected and the cells arelysed using detergents (RIPA buffer, 150 mM NaCl, 1% NP-40, 0.1% SDS,0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culturemedia are precipitated with an HA specific monoclonal antibody.Precipitated polypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the PCIP coding sequence is cloneddirectly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the PCIPpolypeptide is detected by radiolabelling and immunoprecipitation usinga PCIP specific monoclonal antibody.

Rat 1v was cloned into the mammalian expresssion vector pRBG4.Transfections into COS cells were performed using LipofectAmine Plus(Gibco BRL) following the manufacturer's instructions. The expressed 1vprotein was detected by immunocytochemistry and/or western blot analysisusing antibodies raised against 1v in rabbits or mice.

Example 15

Identification and Characterization of Human Full Length P19

The human full length p19 sequence was identified using RACE PCR. Thesequence of p19 (also referred to as KChIP3) is shown in FIG. 16. Theamino acid sequence of human p19 is 92% identical to the mouse p19 gene(SEQ ID NO:35).

TBLASTN searches using the protein sequence of human p19 revealed thathuman p19 is homologous to two sequences, Calsenilin (described in(1998) Nature Medicine 4: 1177-1181) and DREAM, a Ca2+-dependentregulator of prodynorphin and c-fos transcription (described in Carrionet al. (1999) Nature 398: 80-84). Human p1 9 is 100% identical at thenucleotide level to Calsenilin (but extends 3′ to the publishedsequence) and 99% identical at the nucleotide level to DREAM.

The ability of p19 (as well as other PCIP family members) to co-localizewith presenilin and act as transcription factors is determined using artknown techniques such as northern blots, in situ hybridization, β-galassays, DNA mobility assays (described in, for example, Carrion et al.(1999) Nature 398:80) and DNA mobility supershift assays, usingantibodies specific for KchIPs.

Other assays suitable for evaluating the association of PCIP familymembers with presenilins is co-immunoprecipitation (described in, forexample, Buxbaum et al. (1998) Nature Medicine 4:1177).

Example 16

Identification and Characterization of Monkey KChIP4

In this example, the identification and characterization of the genesencoding monkey KChIP4a (jlkbd352e01t1) and alternatively spliced monkeyKChIP4b (jlkbb231c04t1), KChIP4c (jlkxa053c02), and KChIP4d (jlkx015b10)is described. TBLASTN searches in proprietary databases with thesequence of the known PCIP family members, lead to the identification offour clones jlkbb231c04t1, jlkbd352e01t1, jlkxa153c12, and jlkx015b10.The four monkey clones were obtained and sequenced.

The sequences of proprietary monkey clones jlkbb231c04t1 andjlkbd352e01t1 were found to correspond to alternately spliced variantsof an additional PCIP family member, referred to herein as KChIP4. Clonejlkbb231c04t1 contains a 822bp deletion relative to jlkbd352e01t1(presumably due to splicing out of an exon), resulting in the loss ofthe final EF hand domain. In clone jlkbd352e01t1, the final EF handdomain is preserved, and the C-terminus is highly homologous to that ofPCIP family members 1v, 9ql, and p19. Overall identity in the homologousC-termini among KChIP4, 1v, 9ql, and p19 ranged from 71%-80% at theamino acid level (alignments were performed using the CLUSTALW).

Monkey KChIP4c and KChIP4d were discovered by BLASTN search using monkeyKChIP4a as a query for searching a proprietary database.

The nucleotide sequence of the monkey KChIP4a cDNA and the predictedamino acid sequence of the KChIP4a polypeptide are shown in FIG. 23 andin SEQ ID NOs:48 and 49, respectively.

The nucleotide sequence of the monkey KChIP4b cDNA and the predictedamino acid sequence of the KChIP4b polypeptide are shown in FIG. 24 andin SEQ ID NOs:50 and 51, respectively.

The nucleotide sequence of the monkey KChIP4c cDNA and the predictedamino acid sequence of the KChIP4c polypeptide are shown in FIG. 35 andin SEQ ID NOs:69 and 70, respectively.

The nucleotide sequence of the monkey KChIP4d cDNA and the predictedamino acid sequence of the KChIP4d polypeptide are shown in FIG. 36 andin SEQ ID NOs:71 and 72, respectively.

FIG. 37 depicts an alignment of the protein sequences of KChIP4a,KChIP4b, KChIP4c, and KChIP4d.

Rat KChIP4 is predominantly expressed in the brain, and weakly in thekidney, but not in the heart, brain, spleen, lung, liver, skeletalmuscle or testes, as indicated by northern blot experiments in which anorthern blot purchased from Clontech was probed with a DNA fragmentfrom the 3′-untranslated region of rat KChIP4.

Example 17

Identification and Characterization of Human and Rat 33b07

In this example, the identification and characterization of the genesencoding rat and human 33b07 is described. Partial rat 33b07 (clone name9o) was isolated as a positive clone from the yeast two-hybrid screendescribed above, using rKv4.3N as bait. The full length rat 33b07 clonewas identified by mining of proprietary databases.

The nucleotide sequence of the full length rat 33b07 cDNA and thepredicted amino acid sequence of the rat 33b07 polypeptide are shown inFIG. 26 and in SEQ ID NOs:52 and 53, respectively Ihe rat 33b07 cDNAencodes a protein having a molecular weight of approximately 44.7 kD andwhich is 407 amino acid residues in length.

Rat 33b07 binds rKv4.3N and rKv4.2N with slight preference for rKv4.2Nin yeast 2-hybrid assays. In contrast, rat 33b07 does not bind rKv1.1N,indicating that the rat 33b07-Kv4N interaction is specific.

Rat 33b07 is expressed predominantly in the brain as determined bynorthern blot analysis.

The human 33b07 ortholog (clone 106d5) was also identified by mining ofproprietary databases. The nucleotide sequence of the full length human33b07 cDNA and the predicted amino acid sequence of the human 33b07polypeptide are shown in FIG. 27 and in SEQ ID NOs:54 and 55,respectively. The human 33b07 cDNA encodes a protein having a molecularweight of approximately 45.1 kD and which is 414 amino acid residues inlength.

Human 33b07 is 99% identical to the human KIAA0721 protein (GenBankAccession Number: AB018264) at the amino acid level. However, GenBankAccession Number: AB018264 does not have a functional annotation. Human33b07 is also homologous to Testes-specific (Y-encoded) proteins(TSP(Y)s), SET, and Nucleosome Assembly Proteins (NAPs). The human 33b07is 38% identical to human SET protein (GenBank Accession NumberQ01105=U51924) over amino acids 204 to 337 and 46% identical over aminoacids 334 to 387.

Human SET is also called HLA-DR associated protein II (PHAPII)(Hoppe-Seyler (1994) Biol. Chem. 375:113-126) and in some cases isassociated with acute undifferentiated leukemia (AUL) as a result of atranslocation event resulting in the formation of a SET-CAN fusion gene(Von Lindem M. et al. (1992) Mol. Cell. Biol. 12:3346-3355). Analternative spliced form of SET is also called Template ActivatingFactor-I alpha (TAF). TAF is found to be associated with myeloidleukemogenesis (Nagata K. et al. (1995) Proc. Natl. Acad Sci. USA. 92(10), 4279-4283). Human SET is also a potent protein inhibitor ofphosphatase 2A (Adachi Y. et al. (1994) J. Biol. Chem. 269:2258-2262).NAPs may be involved in modulating chromatin formation and contribute toregulation of cell proliferation (Simon H. U. et al. (1994) Biochem. J.297, 389-397).

Thus, due to its homology to the above identified proteins, 33b07 mayfunction as a protein inhibitor of phosphatase, an oncogene, and/or achromatin modulator. The homology of 33b07 to SET, a protein phosphataseinhibitor, is of particular interest. Many channels, in particular theKv4 channels (with which 33b07 is associated), are known to be regulatedby phosphorylation by PKC and PKA ((1998) J. Neuroscience18(10):3521-3528; Am J Physiol 273: H1775-86 (1997)). Thus, 33b07 maymodulate Kv4 activity by regulating the phosphorylation status of thepotassium channel.

Example 18

Identification and Characterization of Rat 1p

In this example, the identification and characterization of the geneencoding rat 1p is described. Partial rat 1p was isolated as a positiveclone from the yeast two-hybrid screen described above, using rKv4.3N asa bait.

The nucleotide sequence of the partial length rat 1p cDNA and thepredicted amino acid sequence of the rat 1p polypeptide are shown inFIG. 28 and in SEQ ID NOs:56 and 57, respectively. The rat 1p cDNAencodes a protein having a molecular weight of approximately 28.6 kD andwhich is 267 amino acid residues in length.

Rat 1p binds rKv4.3N and rKv4.2N with slight preference for rKv4.3N inyeast two-hybrid assays. In contrast, 1p does not bind rKv1.1N,indicating that the 1p-Kv4N interaction is specific.

Rat 1p is predominantly expressed in the brain as determined by northernblot analysis.

A BLASTP 1.4 search, using a score of 100 and a word length of 3(Altschul et al. (1990) J. Mol. Biol. 215:403) of the amino acidsequences of rat 1p revealed that rat 1p is similar to the human Restin(GenBank Accession Number P30622; also named cytoplasmic linkerprotein-170 alpha-2 (CLIP-170), M97501)). The rat 1p protein is 58%identical to the human Restin over amino acid residues 105 to 182, 55%identical to the human Restin over amino acid residues 115 to 186, 22%identical to the human Restin over amino acid residues 173 to 246, 22%identical to the human Restin over amino acid residues 169 to 218, and58% identical to the human Restin over amino acid residues 217 to 228.

Restin is also named Reed-Sternberg intermediate filament associatedprotein. Reed-Sternberg cells are the tumoral cells diagnostic forHodgkin's disease. It is suggested that Restin overexpression may be acontributing factor in the progression of Hodgkin's disease (Bilbe G. etal. (1992) EMBO J. 11:2103-13) and Restin appears to be an intermediatefilament associated protein that links endocytic vesicles tomicrotubules (Pierre P, et al. (1992) Cell 70 (6), 887-900).

The cytoskeleton regulates the activity of potassium channels (see, forexample, Honore E, et al. (1992) EMBO J. 11:2465-2471 and Levin G, etal. (1996) J. Biol. Chem. 271:29321-29328), as well as the activity ofother channels, e.g., Ca⁺⁺ channels (Johnson B. D. et al. (1993) Neuron10:797-804); or Na⁺ channels (Fukuda J. et al. (1981) Nature 294:82-85).

Accordingly, based on its homology to the Restin protein, the rat 1pprotein may be associated with the cytoskeleton and may modulate theactivity of potassium channels, e.g., Kv4, via its association to thecytoskeleton.

Example 19

Identification and Characterization of Rat 7s

In this example, the identification and characterization of the geneencoding rat 7s is described. Partial rat 7s was isolated as a positiveclone from the yeast two-hybrid screen described above, using rKv4.3N asa bait. Rat 7s is the rat ortholog of the human vacuolar H(+)-ATPasecatalytic subunit A (Accession Number P38606 and B46091) described in,for example, van Hille B. et al. (1993) J. Biol. Chem. 268 (10),7075-7080.

The nucleotide sequence of the partial length rat 7s cDNA and thepredicted amino acid sequence of the rat 7s polypeptide are shown inFIG. 29 and in SEQ ID NOs:58 and 59, respectively. The rat 7s cDNAencodes a protein having a molecular weight of approximately 28.6 kD andwhich is 270 amino acid residues in length.

Rat 7s binds rKv4.3N and rKv4.2N with preference for rKv4.3N in yeasttwo-hybrid assays. In contrast, 7s does not bind rKv1.1N, indicatingthat the 7s-Kv4N interaction is specific.

Rat 7s is expressed at significantly higher levels in the brain and thekidney than in the lung, liver, heart, testes, and skeletal muscle, asdetermined by northern blot analysis.

Example 20

Identification and Characterization of Rat 29x and 25r

In this example, the identification and characterization of the geneencoding rat 29x is described. Rat 29x was isolated as a positive clonefrom the yeast two-hybrid screen described above, using rKv4.3N as abait. Rat 25r is a splice variant of 29x. They differ in the 5′untranslated region, but are identical in the coding region and at theamino acid level.

The nucleotide sequence of the rat 29x cDNA and the predicted amino acidsequence of the rat 29x polypeptide are shown in FIG. 30 and in SEQ IDNOs:60 and 61, respectively. The rat 29x cDNA encodes a protein having amolecular weight of approximately 40.4 kD and which is 351 amino acidresidues in length.

The nucleotide sequence of the rat 25r cDNA is shown in FIG. 31 and inSEQ ID NO:62. The rat 25r cDNA encodes a protein having a molecularweight of approximately 40.4 kD and which is 351 amino acid residues inlength.

Rat 29x is expressed in the spleen, lung, kidney, heart, brain, testes,skeletal muscle and liver, with the highest level of expression being inthe spleen and the lowest being in the liver.

Rat 29x binds rKv4.3N and rKv4.2N with slight preference for rKv4.3N inyeast two-hybrid assays. In contrast, 29x does not bind rKv1.1N,indicating that the 29x-Kv4N interaction is specific.

Rat 29x is identical at the amino acid level to rat SOCS-1 (SuppressorOf Cytokine Signaling) described in Starr R. et al. (1997) Nature 387:917-921; to JAB described in Endo T. A. et al. (1997) Nature 387:921-924; and to SSI-1 (STAT-induced STAT inhibitor-1) described in NakaT. et al. (1997) Nature 387:924-928. These proteins are characterized inthat they have an SH2 domain, bind to and inhibit JAK kinase, and, as aresult, regulate cytokine signaling.

As used herein, the term “SH2 domain”, also referred to a Src Homology 2domain, includes a protein domain of about 100 amino acids in lengthwhich is involved in binding of phosphotyrosine residues, e.g.,phosphotyrosine residues in other proteins. The target site is called anSH2-binding site. The SH2 domain has a conserved 3D structure consistingof two alpha helices and six to seven beta-strands. The core of the SH2domain is formed by a continuous beta-meander composed of two connectedbeta-sheets (Kuriyan J. et al. (1997) Curr. Opin. Struct. Biol.3:828-837). SH2 domains function as regulatory modules of intracellularsignaling cascades by interacting with high affinity tophosphotyrosine-containing target peptides in a sequence-specific andstrictly phosphorylation-dependent manner (Pawson T. (1995) Nature373:573-580). Some proteins contain multiple SH2 domains, whichincreases their affinity for binding to phosphoproteins or confers theability to bind to different phosphoproteins. Rat 29x contains an SH2domain at amino acid residues 219-308 of SEQ ID NO:61.

Tyrosine phosphorylation regulates potassium channel activity(Prevarskaya N. B. et al. (1995) J. BioL Chem. 270:24292-24299). JAKkinase phoshorylates proteins at tyrosines and is implicated in theregulation of channel activity (Prevarskaya N. B. et al. supra).Accordingly, based on its homology to SOCS-1, JAB, and SSI-1, rat 29xmay modulate the activity of potassium channels, e.g., Kv4, bymodulating JAK kinase activity.

Example 21

Identification and Characterization of Rat 5p

In this example, the identification and characterization of the geneencoding rat 5p is described. Rat 5p was isolated as a positive clonefrom the yeast two-hybrid screen described above, using rKv4.3N as abait.

The nucleotide sequence of the rat 5pc DNA and the predicted amino acidsequence of the rat 5p polypeptide are shown in FIG. 32 and in SEQ IDNOs:63 and 64, respectively. The rat 5p cDNA encodes a protein having amolecular weight of approximately 11.1 kD and which is 95 amino acidresidues in length.

Rat 5p binds rKv4.3N and rKv4.2N with similar strength in yeasttwo-hybrid assays. In contrast, 5p does not bind rKv1.1N, indicatingthat the 5p-Kv4N interaction is specific.

Rat 5p is expressed in the spleen, lung, skeletal muscle, heart, kidney,brain, liver, and testes, as determined by northern blot analysis.

The rat 5p is identical to rat Calpactin I light chain or P10 (AccessionNumber P05943). P10 binds and induces the dimerization of annexin II(p36). P10 may function as a regulator of protein phosphorylation inthat the p36 monomer is the preferred target of a tyrosine-specifickinase (Masiakowski P. et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 85(4): 1277-1281).

Tyrosine phosphorylation regulates the activity of potassium channels(Prevarskaya N. B. et al. supra). Thus, due to its identity to P10, rat5p may modulate the activity of potassium channels, e.g., Kv4, bymodulating the activity of a tyrosine-specific kinase.

Example 22

Identification and Characterization of Rat 7q

In this example, the identification and characterization of the geneencoding rat 7q is described. Rat 7q was isolated as a positive clonefrom the yeast two-hybrid screen described above, using rKv4.3N as abait. Full length rat 7q was obtained by RACE PCR.

The nucleotide sequence of the rat 7q cDNA and the predicted amino acidsequence of the rat 7q polypeptide are shown in FIG. 33 and in SEQ IDNOs:65 and 66, respectively. The rat 7q cDNA encodes a protein having amolecular weight of approximately 23.5 kD and which is 212 amino acidresidues in length.

Rat 7q binds rKv4.3N and rKv4.2N with same strength in yeast two-hybridassays. In contrast, 7q does not bind rKv1.1N indicating that the7q-Kv4N interaction is specific.

Rat 7q is expressed in the heart, brain, spleen, lung, liver, skeletalmuscle, kidney, and testes, as determined by northern blot analysis.

Rat 7q is identical to RAB2 (rat RAS-related protein, Accession NumberP05712) at the amino acid level. RAB2 appears to be involved invesicular traffic and protein transport (Touchot N. et al. (1987) Proc.Natl. Acad. Sci. US.A. 84 (23):8210-8214). Accordingly, based on itshomology to RAB2, rat 7q may be involved in potassium channel, e.g.,Kv4, trafficking.

Example 23

Identification and Characterization of Rat 19r

In this example, the identification and characterization of the geneencoding rat 19r is described. Partial rat 19r was isolated as apositive clone from the yeast two-hybrid screen described above, usingrKv4.3N as a bait. Full length rat 19r was obtained by RACE PCR.

The nucleotide sequence of the rat 19r cDNA and the predicted amino acidsequence of the rat 19r polypeptide are shown in FIG. 34 and in SEQ IDNOs:67 and 68, respectively. The rat 19r cDNA encodes a protein having amolecular weight of approximately 31.9 kD and which is 271 amino acidresidues in length.

Rat 19r is expressed in the heart, brain, spleen, lung, liver, skeletalmuscle, kidney, and testes, as determined by northern blot analysis.

Rat 19r binds rKv4.3N and rKv4.2N with slight preference for rKv4.3N inyeast two-hybrid assays. In contrast, 19r does not bind rKv1.1N,indicating that the 19r-Kv4N interaction is specific.

Rat 19r is ical to Rat phosphatidylinositol (PTDINS) transfer proteinalpha (PTDINSTP, Accession Number M25758 or P16446) described inDickeson S. K. et al. (1989) J. Biol. Chem. 264:16557-16564. PTDINSTP isbelieved to be involved in phospholipase C-beta (PLC-beta) signaling,phosphatidylinositol transfer protein (PtdIns-TP) synthesis, secrettoryvesicle formation, and enhancement of phosphatidylinositol 3-kinase(PtdIns 3-kinase) activity (Cunningham E. et al. (1995) Curr. Biol.(7):775-783; (1995) Nature 377 (6549):544-547; and Panaretou C. et al.(1997) J. Biol. Chem. 272 (4): 2477-2485).

Accordingly, based on its homology with PTDINSTP, rat 19r may modulatepotassium channel, e.g., Kv4, activity via the PLC-beta signalingpathway and/or the PtdIns 3-kinase signaling pathway. Rat p19r may alsobe involved in potassium channel, e.g., Kv4, trafficking.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

73 1 1463 DNA Homo sapiens CDS (225)..(872) 1 gaatagcccc ctttcacttctgagtccctg catgtgcggg gctgaagaag gaagccagaa 60 gcctcctagc ctcgcctccacgtttgctga ataccaagct gcaggcgagc tgccgggcgc 120 ttttctctcc tccaattcagagtagacaaa ccacggggat ttctttccag ggtaggggag 180 gggccgggcc cggggtcccaactcgcactc aagtcttcgc tgcc atg ggg gcc gtc 236 Met Gly Ala Val 1 atg ggcacc ttc tca tct ctg caa acc aaa caa agg cga ccc tcg aaa 284 Met Gly ThrPhe Ser Ser Leu Gln Thr Lys Gln Arg Arg Pro Ser Lys 5 10 15 20 gat aagatt gaa gat gag ctg gag atg acc atg gtt tgc cat cgg ccc 332 Asp Lys IleGlu Asp Glu Leu Glu Met Thr Met Val Cys His Arg Pro 25 30 35 gag gga ctggag cag ctc gag gcc cag acc aac ttc acc aag agg gag 380 Glu Gly Leu GluGln Leu Glu Ala Gln Thr Asn Phe Thr Lys Arg Glu 40 45 50 ctg cag gtc ctttat cga ggc ttc aaa aat gag tgc ccc agt ggt gtg 428 Leu Gln Val Leu TyrArg Gly Phe Lys Asn Glu Cys Pro Ser Gly Val 55 60 65 gtc aac gaa gac acattc aag cag atc tat gct cag ttt ttc cct cat 476 Val Asn Glu Asp Thr PheLys Gln Ile Tyr Ala Gln Phe Phe Pro His 70 75 80 gga gat gcc agc acg tatgcc cat tac ctc ttc aat gcc ttc gac acc 524 Gly Asp Ala Ser Thr Tyr AlaHis Tyr Leu Phe Asn Ala Phe Asp Thr 85 90 95 100 act cag aca ggc tcc gtgaag ttc gag gac ttt gta acc gct ctg tcg 572 Thr Gln Thr Gly Ser Val LysPhe Glu Asp Phe Val Thr Ala Leu Ser 105 110 115 att tta ttg aga gga actgtc cac gag aaa cta agg tgg aca ttt aat 620 Ile Leu Leu Arg Gly Thr ValHis Glu Lys Leu Arg Trp Thr Phe Asn 120 125 130 ttg tat gac atc aac aaggac gga tac ata aac aaa gag gag atg atg 668 Leu Tyr Asp Ile Asn Lys AspGly Tyr Ile Asn Lys Glu Glu Met Met 135 140 145 gac att gtc aaa gcc atctat gac atg atg ggg aaa tac aca tat cct 716 Asp Ile Val Lys Ala Ile TyrAsp Met Met Gly Lys Tyr Thr Tyr Pro 150 155 160 gtg ctc aaa gag gac actcca agg cag cat gtg gac gtc ttc ttc cag 764 Val Leu Lys Glu Asp Thr ProArg Gln His Val Asp Val Phe Phe Gln 165 170 175 180 aaa atg gac aaa aataaa gat ggc atc gta act tta gat gaa ttt ctt 812 Lys Met Asp Lys Asn LysAsp Gly Ile Val Thr Leu Asp Glu Phe Leu 185 190 195 gaa tca tgt cag gaggac gac aac atc atg agg tct ctc cag ctg ttt 860 Glu Ser Cys Gln Glu AspAsp Asn Ile Met Arg Ser Leu Gln Leu Phe 200 205 210 caa aat gtc atgtaactggtga cactcagcca ttcagctctc agagacattg 912 Gln Asn Val Met 215tactaaacaa ccaccttaac accctgatct gcccttgttc tgattttaca caccaactct 972tgggacagaa acacctttta cactttggaa gaattctctg ctgaagactt tcttatggaa 1032cccagcatca tgtggctcag tctctgattg ccaactcttc ctctttcttc ttcttgagag 1092agacaagatg aaatttgagt ttgttttgga agcatgctca tctcctcaca ctgctgccct 1152atggaaggtc cctctgctta agcttaaaca gtagtgcaca aaatatgctg cttacgtgcc 1212cccagcccac tgcctccaag tcaggcagac cttggtgaat ctggaagcaa gaggacctga 1272gccagatgca caccatctct gatggcctcc caaaccaatg tgcctgtttc tcttcctttg 1332gtgggaagaa tgagagttat ccagaacaat taggatctgt catgaccaga ttgggagagc 1392cagcacctaa catatgtggg ataggactga attattaagc atgacattgt ctgatgaccc 1452aaactgcccc g 1463 2 216 PRT Homo sapiens 2 Met Gly Ala Val Met Gly ThrPhe Ser Ser Leu Gln Thr Lys Gln Arg 1 5 10 15 Arg Pro Ser Lys Asp LysIle Glu Asp Glu Leu Glu Met Thr Met Val 20 25 30 Cys His Arg Pro Glu GlyLeu Glu Gln Leu Glu Ala Gln Thr Asn Phe 35 40 45 Thr Lys Arg Glu Leu GlnVal Leu Tyr Arg Gly Phe Lys Asn Glu Cys 50 55 60 Pro Ser Gly Val Val AsnGlu Asp Thr Phe Lys Gln Ile Tyr Ala Gln 65 70 75 80 Phe Phe Pro His GlyAsp Ala Ser Thr Tyr Ala His Tyr Leu Phe Asn 85 90 95 Ala Phe Asp Thr ThrGln Thr Gly Ser Val Lys Phe Glu Asp Phe Val 100 105 110 Thr Ala Leu SerIle Leu Leu Arg Gly Thr Val His Glu Lys Leu Arg 115 120 125 Trp Thr PheAsn Leu Tyr Asp Ile Asn Lys Asp Gly Tyr Ile Asn Lys 130 135 140 Glu GluMet Met Asp Ile Val Lys Ala Ile Tyr Asp Met Met Gly Lys 145 150 155 160Tyr Thr Tyr Pro Val Leu Lys Glu Asp Thr Pro Arg Gln His Val Asp 165 170175 Val Phe Phe Gln Lys Met Asp Lys Asn Lys Asp Gly Ile Val Thr Leu 180185 190 Asp Glu Phe Leu Glu Ser Cys Gln Glu Asp Asp Asn Ile Met Arg Ser195 200 205 Leu Gln Leu Phe Gln Asn Val Met 210 215 3 1856 DNA Rattussp. CDS (300)..(1034) 3 ggcacacaac ccctggattc ttcggagaat atgccgtgaggtgttgccaa ttattagttc 60 tcttggctag cagatgttta gggactggtt aagcctttggagaaattacc ttaggaaaac 120 ggggaaataa aagcaaagat taccatgaat tgcaagattacctagcaatt gcaaggtagg 180 aggagagagg tggagggcgg agtagacagg agggagggagaaagtgagag gaagctaggc 240 tggtggaaat aaccctgcac ttggaacagc ggcaaagaagcgcgattttc cagctttaa 299 atg cct gcc cgc gtt ctg ctt gcc tac ccg gga acggag atg ttg acc 347 Met Pro Ala Arg Val Leu Leu Ala Tyr Pro Gly Thr GluMet Leu Thr 1 5 10 15 cag ggc gag tct gaa ggg ctc cag acc ttg ggg atagta gtg gtc ctg 395 Gln Gly Glu Ser Glu Gly Leu Gln Thr Leu Gly Ile ValVal Val Leu 20 25 30 tgt tcc tct ctg aaa cta ctg cac tac ctc ggg ctg attgac ttg tcg 443 Cys Ser Ser Leu Lys Leu Leu His Tyr Leu Gly Leu Ile AspLeu Ser 35 40 45 gat gac aag atc gag gat gat ctg gag atg acc atg gtt tgccat cgg 491 Asp Asp Lys Ile Glu Asp Asp Leu Glu Met Thr Met Val Cys HisArg 50 55 60 cct gag gga ctg gag cag ctt gag gca cag acg aac ttc acc aagaga 539 Pro Glu Gly Leu Glu Gln Leu Glu Ala Gln Thr Asn Phe Thr Lys Arg65 70 75 80 gaa ctg caa gtc ctt tac cgg gga ttc aaa aac gag tgc ccc agtggt 587 Glu Leu Gln Val Leu Tyr Arg Gly Phe Lys Asn Glu Cys Pro Ser Gly85 90 95 gtg gtt aac gaa gag aca ttc aag cag atc tac gct cag ttt ttc cct635 Val Val Asn Glu Glu Thr Phe Lys Gln Ile Tyr Ala Gln Phe Phe Pro 100105 110 cat gga gat gcc agc aca tac gca cat tac ctc ttc aat gcc ttc gac683 His Gly Asp Ala Ser Thr Tyr Ala His Tyr Leu Phe Asn Ala Phe Asp 115120 125 acc acc cag aca ggc tct gta aag ttc gag gac ttt gtg act gct ctg731 Thr Thr Gln Thr Gly Ser Val Lys Phe Glu Asp Phe Val Thr Ala Leu 130135 140 tcg att tta ctg aga gga acg gtc cat gaa aaa ctg agg tgg acg ttt779 Ser Ile Leu Leu Arg Gly Thr Val His Glu Lys Leu Arg Trp Thr Phe 145150 155 160 aat ttg tac gac atc aat aaa gac ggc tac ata aac aaa gag gagatg 827 Asn Leu Tyr Asp Ile Asn Lys Asp Gly Tyr Ile Asn Lys Glu Glu Met165 170 175 atg gac ata gtg aaa gcc atc tat gac atg atg ggg aaa tac acctat 875 Met Asp Ile Val Lys Ala Ile Tyr Asp Met Met Gly Lys Tyr Thr Tyr180 185 190 cct gtg ctc aaa gag gac act ccc agg cag cac gtg gac gtc ttcttc 923 Pro Val Leu Lys Glu Asp Thr Pro Arg Gln His Val Asp Val Phe Phe195 200 205 cag aaa atg gat aaa aat aaa gat ggc att gta acg tta gac gaattt 971 Gln Lys Met Asp Lys Asn Lys Asp Gly Ile Val Thr Leu Asp Glu Phe210 215 220 ctc gag tcc tgt cag gag gat gac aac atc atg agg tct cta cagctg 1019 Leu Glu Ser Cys Gln Glu Asp Asp Asn Ile Met Arg Ser Leu Gln Leu225 230 235 240 ttc caa aat gtc atg taactgagga cactggccat cctgctctcagagacactga 1074 Phe Gln Asn Val Met 245 caaacacctc aatgccctga tctgcccttgttccagtttt acacatcaac tctcgggaca 1134 gaaatacctt ttacactttg gaagaattctctgctgaaga ctttctacaa aacctggcac 1194 cgagtggctc agtctctgat tgccaactcttcctccctcc tcctcttgag agggacgagc 1254 tgaaatccga agtttgtttt ggaagcatgcccatctctcc atgctgctgc tgccctgtgg 1314 aaggcccctc tgcttgagct taaacagtagtgcacagttt tctgcgtata cagatcccca 1374 actcactgcc tctaagtcag gcagaccctgatcaatctga accaaatgtg caccatcctc 1434 cgatggcctc ccaagccaat gtgcctgcttctcttcctct ggtgggaaga aagaacgctc 1494 tacagagcac ttagagctta ccatgaaaatactgggagag gcagcaccta acacatgtag 1554 aataggactg aattattaag catggtggtatcagatgatg caaacagccc atgtcatttt 1614 tttttccaga ggtagggact aataattctcccacactagc acctacgatc atagaacaag 1674 tcttttaaca catccaggag ggaaaccgctgcccagtggt ctatcccttc tctccatccc 1734 ctgctcaagc ccagcactgc atgtctctcccggaaggtcc agaatgcctg tgaaatgctg 1794 taacttttat accctgttat aatcaataaacagaactatt tcgtacaaaa aaaaaaaaaa 1854 aa 1856 4 245 PRT Rattus sp. 4 MetPro Ala Arg Val Leu Leu Ala Tyr Pro Gly Thr Glu Met Leu Thr 1 5 10 15Gln Gly Glu Ser Glu Gly Leu Gln Thr Leu Gly Ile Val Val Val Leu 20 25 30Cys Ser Ser Leu Lys Leu Leu His Tyr Leu Gly Leu Ile Asp Leu Ser 35 40 45Asp Asp Lys Ile Glu Asp Asp Leu Glu Met Thr Met Val Cys His Arg 50 55 60Pro Glu Gly Leu Glu Gln Leu Glu Ala Gln Thr Asn Phe Thr Lys Arg 65 70 7580 Glu Leu Gln Val Leu Tyr Arg Gly Phe Lys Asn Glu Cys Pro Ser Gly 85 9095 Val Val Asn Glu Glu Thr Phe Lys Gln Ile Tyr Ala Gln Phe Phe Pro 100105 110 His Gly Asp Ala Ser Thr Tyr Ala His Tyr Leu Phe Asn Ala Phe Asp115 120 125 Thr Thr Gln Thr Gly Ser Val Lys Phe Glu Asp Phe Val Thr AlaLeu 130 135 140 Ser Ile Leu Leu Arg Gly Thr Val His Glu Lys Leu Arg TrpThr Phe 145 150 155 160 Asn Leu Tyr Asp Ile Asn Lys Asp Gly Tyr Ile AsnLys Glu Glu Met 165 170 175 Met Asp Ile Val Lys Ala Ile Tyr Asp Met MetGly Lys Tyr Thr Tyr 180 185 190 Pro Val Leu Lys Glu Asp Thr Pro Arg GlnHis Val Asp Val Phe Phe 195 200 205 Gln Lys Met Asp Lys Asn Lys Asp GlyIle Val Thr Leu Asp Glu Phe 210 215 220 Leu Glu Ser Cys Gln Glu Asp AspAsn Ile Met Arg Ser Leu Gln Leu 225 230 235 240 Phe Gln Asn Val Met 2455 1907 DNA Mus musculus CDS (477)..(1124) 5 cggccccctg agatccagcccgagcgcggg gcggagcggc cgggtggcag caggggcggg 60 cgggcggagc gcagctcccgcaccgcacgc ggcgcgggct cggcagcctc ggccgtgcgg 120 gcacgccggc cccgtgtccaacatcaggca ggctttgggg ctcggggctc gggcctcgga 180 gaagccagtg gcccggctgggtgcccgcac cggggggcgc ctgtgaaggc tcccgcgagc 240 ctctggccct gggagtcagtgcatgtgcct ggctgaagaa ggcagcagcc acgagctcca 300 ggcgccccgg ccccacgttttctgaatacc aagctgcagg cgagctgctc ggggcttttt 360 tgctttctcg cttttcctctcctccaattc aaagtgggca atccacaccg atttcttttc 420 aggggaggga agagacagggcctggggtcc caagacgcac acaagtcttc gctgcc atg 479 Met 1 ggg gcc gtc atgggc act ttc tcc tcc ctg cag acc aaa caa agg cga 527 Gly Ala Val Met GlyThr Phe Ser Ser Leu Gln Thr Lys Gln Arg Arg 5 10 15 ccc tct aaa gac aagatt gag gat gag cta gag atg acc atg gtt tgc 575 Pro Ser Lys Asp Lys IleGlu Asp Glu Leu Glu Met Thr Met Val Cys 20 25 30 cac cgg cct gag gga ctggag cag ctt gag gca cag acg aac ttc acc 623 His Arg Pro Glu Gly Leu GluGln Leu Glu Ala Gln Thr Asn Phe Thr 35 40 45 aag aga gaa ctg caa gtc ttgtac cgg gga ttc aaa aac gag tgc cct 671 Lys Arg Glu Leu Gln Val Leu TyrArg Gly Phe Lys Asn Glu Cys Pro 50 55 60 65 agc ggt gtg gtc aat gaa gaaaca ttc aag cag atc tac gct cag ttt 719 Ser Gly Val Val Asn Glu Glu ThrPhe Lys Gln Ile Tyr Ala Gln Phe 70 75 80 ttc cct cac gga gat gcc agc acatat gca cat tac ctc ttc aat gcc 767 Phe Pro His Gly Asp Ala Ser Thr TyrAla His Tyr Leu Phe Asn Ala 85 90 95 ttc gac acc acc cag aca ggc tct gtaaag ttc gag gac ttt gtg act 815 Phe Asp Thr Thr Gln Thr Gly Ser Val LysPhe Glu Asp Phe Val Thr 100 105 110 gct ctg tcg att tta ctg aga ggg acagtc cat gaa aaa cta agg tgg 863 Ala Leu Ser Ile Leu Leu Arg Gly Thr ValHis Glu Lys Leu Arg Trp 115 120 125 acg ttt aat ttg tat gac atc aat aaagac ggc tac ata aac aaa gag 911 Thr Phe Asn Leu Tyr Asp Ile Asn Lys AspGly Tyr Ile Asn Lys Glu 130 135 140 145 gag atg atg gac ata gtc aaa gccatc tat gac atg atg ggg aaa tac 959 Glu Met Met Asp Ile Val Lys Ala IleTyr Asp Met Met Gly Lys Tyr 150 155 160 acc tat cct gtg ctc aaa gag gacact ccc agg cag cat gtg gat gtc 1007 Thr Tyr Pro Val Leu Lys Glu Asp ThrPro Arg Gln His Val Asp Val 165 170 175 ttc ttc cag aaa atg gat aaa aataaa gat ggc att gta acg tta gat 1055 Phe Phe Gln Lys Met Asp Lys Asn LysAsp Gly Ile Val Thr Leu Asp 180 185 190 gaa ttt ctt gaa tca tgt cag gaggat gac aac atc atg aga tct cta 1103 Glu Phe Leu Glu Ser Cys Gln Glu AspAsp Asn Ile Met Arg Ser Leu 195 200 205 cag ctg ttc caa aat gtc atgtaactgagga cactggccat tctgctctca 1154 Gln Leu Phe Gln Asn Val Met 210215 gagacactga caaacacctt aatgccctga tctgcccttg ttccaatttt acacaccaac1214 tcttgggaca gaaatacctt ttacactttg gaagaattct ctgctgaaga ctttctacaa1274 aacctggcac cacgtggctc tgtctctgag ggacgagcgg agatccgact ttgttttgga1334 agcatgccca tctcttcatg ctgctgccct gtggaaggcc cctctgcttg agcttaatca1394 atagtgcaca gttttatgct tacacatatc cccaactcac tgcctccaag tcaggcagac1454 tctgatgaat ctgagccaaa tgtgcaccat cctccgatgg cctcccaagc caatgtgcct1514 gcttctcttc ctctggtggg aagaaagagt gttctacgga acaattagag cttaccatga1574 aaatattggg agaggcagca cctaacacat gtagaatagg actgaattat taagcatggt1634 gatatcagat gatgcaaatt gcccatgtca tttttttcaa aggtagggac aaatgattct1694 cccacactag cacctgtggt catagagcaa gtctcttaac atgcccagaa ggggaaccac1754 tgtccagtgg tctatccctc ctctccatcc cctgctcaaa cccagcactg catgtccctc1814 caagaaggtc cagaatgcct gcgaaacgct gtacttttat accctgttct aatcaataaa1874 cagaactatt tcgtaaaaaa aaaaaaaaaa aaa 1907 6 216 PRT Mus musculus 6Met Gly Ala Val Met Gly Thr Phe Ser Ser Leu Gln Thr Lys Gln Arg 1 5 1015 Arg Pro Ser Lys Asp Lys Ile Glu Asp Glu Leu Glu Met Thr Met Val 20 2530 Cys His Arg Pro Glu Gly Leu Glu Gln Leu Glu Ala Gln Thr Asn Phe 35 4045 Thr Lys Arg Glu Leu Gln Val Leu Tyr Arg Gly Phe Lys Asn Glu Cys 50 5560 Pro Ser Gly Val Val Asn Glu Glu Thr Phe Lys Gln Ile Tyr Ala Gln 65 7075 80 Phe Phe Pro His Gly Asp Ala Ser Thr Tyr Ala His Tyr Leu Phe Asn 8590 95 Ala Phe Asp Thr Thr Gln Thr Gly Ser Val Lys Phe Glu Asp Phe Val100 105 110 Thr Ala Leu Ser Ile Leu Leu Arg Gly Thr Val His Glu Lys LeuArg 115 120 125 Trp Thr Phe Asn Leu Tyr Asp Ile Asn Lys Asp Gly Tyr IleAsn Lys 130 135 140 Glu Glu Met Met Asp Ile Val Lys Ala Ile Tyr Asp MetMet Gly Lys 145 150 155 160 Tyr Thr Tyr Pro Val Leu Lys Glu Asp Thr ProArg Gln His Val Asp 165 170 175 Val Phe Phe Gln Lys Met Asp Lys Asn LysAsp Gly Ile Val Thr Leu 180 185 190 Asp Glu Phe Leu Glu Ser Cys Gln GluAsp Asp Asn Ile Met Arg Ser 195 200 205 Leu Gln Leu Phe Gln Asn Val Met210 215 7 1534 DNA Rattus sp. CDS (31)..(711) 7 gtcccaagtc gcacacaagtcttcgctgcc atg ggg gcc gtc atg ggt acc ttc 54 Met Gly Ala Val Met GlyThr Phe 1 5 tcg tcc ctg cag acc aaa caa agg cga ccc tct aaa gac atc gcctgg 102 Ser Ser Leu Gln Thr Lys Gln Arg Arg Pro Ser Lys Asp Ile Ala Trp10 15 20 tgg tat tac cag tat cag aga gac aag atc gag gat gat ctg gag atg150 Trp Tyr Tyr Gln Tyr Gln Arg Asp Lys Ile Glu Asp Asp Leu Glu Met 2530 35 40 acc atg gtt tgc cat cgg cct gag gga ctg gag cag ctt gag gca cag198 Thr Met Val Cys His Arg Pro Glu Gly Leu Glu Gln Leu Glu Ala Gln 4550 55 acg aac ttc acc aag aga gaa ctg caa gtc ctt tac cgg gga ttc aaa246 Thr Asn Phe Thr Lys Arg Glu Leu Gln Val Leu Tyr Arg Gly Phe Lys 6065 70 aac gag tgc ccc agt ggt gtg gtt aac gaa gag aca ttc aag cag atc294 Asn Glu Cys Pro Ser Gly Val Val Asn Glu Glu Thr Phe Lys Gln Ile 7580 85 tac gct cag ttt ttc cct cat gga gat gcc agc aca tac gca cat tac342 Tyr Ala Gln Phe Phe Pro His Gly Asp Ala Ser Thr Tyr Ala His Tyr 9095 100 ctc ttc aat gcc ttc gac acc acc cag aca ggc tct gta aag ttc gag390 Leu Phe Asn Ala Phe Asp Thr Thr Gln Thr Gly Ser Val Lys Phe Glu 105110 115 120 gac ttt gtg act gct ctg tcg att tta ctg aga gga acg gtc catgaa 438 Asp Phe Val Thr Ala Leu Ser Ile Leu Leu Arg Gly Thr Val His Glu125 130 135 aaa ctg agg tgg acg ttt aat ttg tac gac atc aat aaa gac ggctac 486 Lys Leu Arg Trp Thr Phe Asn Leu Tyr Asp Ile Asn Lys Asp Gly Tyr140 145 150 ata aac aaa gag gag atg atg gac ata gtg aaa gcc atc tat gacatg 534 Ile Asn Lys Glu Glu Met Met Asp Ile Val Lys Ala Ile Tyr Asp Met155 160 165 atg ggg aaa tac acc tat cct gtg ctc aaa gag gac act ccc aggcag 582 Met Gly Lys Tyr Thr Tyr Pro Val Leu Lys Glu Asp Thr Pro Arg Gln170 175 180 cac gtg gac gtc ttc ttc cag aaa atg gat aaa aat aaa gat ggcatt 630 His Val Asp Val Phe Phe Gln Lys Met Asp Lys Asn Lys Asp Gly Ile185 190 195 200 gta acg tta gac gaa ttt ctc gag tcc tgt cag gag gat gacaac atc 678 Val Thr Leu Asp Glu Phe Leu Glu Ser Cys Gln Glu Asp Asp AsnIle 205 210 215 atg agg tct cta cag ctg ttc caa aat gtc atg taactgaggacactggccat 731 Met Arg Ser Leu Gln Leu Phe Gln Asn Val Met 220 225cctgctctca gagacactga caaacacctc aatgccctga tctgcccttg ttccagtttt 791acacatcaac tctcgggaca gaaatacctt ttacactttg gaagaattct ctgctgaaga 851ctttctacaa aacctggcac cgcgtggctc agtctctgat tgccaactct tcctccctcc 911tcctcttgag agggacgagc tgaaatccga agtttgtttt ggaagcatgc ccatctctcc 971atgctgctgc tgccctgtgg aaggcccctc tgcttgagct taaacagtag tgcacagttt 1031tctgcgtata cagatcccca actcactgcc tctaagtcag gcagaccctg atcaatctga 1091accaaatgtg caccatcctc cgatggcctc ccaagccaat gtgcctgctt ctcttcctct 1151ggtgggaaga aagaacgctc tacagagcac ttagagctta ccatgaaaat actgggagag 1211gcagcaccta acacatgtag aataggactg aattattaag catggtggta tcagatgatg 1271caaacagccc atgtcatttt ttttccagag gtagggacta ataattctcc cacactagca 1331cctacgatca tagaacaagt cttttaacac atccaggagg gaaaccgctg cccagtggtc 1391tatcccttct ctccatcccc tgctcaagcc cagcactgca tgtctctccc ggaaggtcca 1451gaatgcctgt gaaatgctgt aacttttata ccctgttata atcaataaac agaactattt 1511cgtacaaaaa aaaaaaaaaa aaa 1534 8 227 PRT Rattus sp. 8 Met Gly Ala ValMet Gly Thr Phe Ser Ser Leu Gln Thr Lys Gln Arg 1 5 10 15 Arg Pro SerLys Asp Ile Ala Trp Trp Tyr Tyr Gln Tyr Gln Arg Asp 20 25 30 Lys Ile GluAsp Asp Leu Glu Met Thr Met Val Cys His Arg Pro Glu 35 40 45 Gly Leu GluGln Leu Glu Ala Gln Thr Asn Phe Thr Lys Arg Glu Leu 50 55 60 Gln Val LeuTyr Arg Gly Phe Lys Asn Glu Cys Pro Ser Gly Val Val 65 70 75 80 Asn GluGlu Thr Phe Lys Gln Ile Tyr Ala Gln Phe Phe Pro His Gly 85 90 95 Asp AlaSer Thr Tyr Ala His Tyr Leu Phe Asn Ala Phe Asp Thr Thr 100 105 110 GlnThr Gly Ser Val Lys Phe Glu Asp Phe Val Thr Ala Leu Ser Ile 115 120 125Leu Leu Arg Gly Thr Val His Glu Lys Leu Arg Trp Thr Phe Asn Leu 130 135140 Tyr Asp Ile Asn Lys Asp Gly Tyr Ile Asn Lys Glu Glu Met Met Asp 145150 155 160 Ile Val Lys Ala Ile Tyr Asp Met Met Gly Lys Tyr Thr Tyr ProVal 165 170 175 Leu Lys Glu Asp Thr Pro Arg Gln His Val Asp Val Phe PheGln Lys 180 185 190 Met Asp Lys Asn Lys Asp Gly Ile Val Thr Leu Asp GluPhe Leu Glu 195 200 205 Ser Cys Gln Glu Asp Asp Asn Ile Met Arg Ser LeuGln Leu Phe Gln 210 215 220 Asn Val Met 225 9 1540 DNA Mus musculus CDS(77)..(757) 9 atccacaccg atttcttttc aggggaggga agagacaggg cctggggtcccaagacgcac 60 acaagtcttc gctgcc atg ggg gcc gtc atg ggc act ttc tcc tccctg cag 112 Met Gly Ala Val Met Gly Thr Phe Ser Ser Leu Gln 1 5 10 accaaa caa agg cga ccc tct aaa gac atc gcc tgg tgg tat tac cag 160 Thr LysGln Arg Arg Pro Ser Lys Asp Ile Ala Trp Trp Tyr Tyr Gln 15 20 25 tat cagaga gac aag att gag gat gag cta gag atg acc atg gtt tgc 208 Tyr Gln ArgAsp Lys Ile Glu Asp Glu Leu Glu Met Thr Met Val Cys 30 35 40 cac cgg cctgag gga ctg gag cag ctt gag gca cag acg aac ttc acc 256 His Arg Pro GluGly Leu Glu Gln Leu Glu Ala Gln Thr Asn Phe Thr 45 50 55 60 aag aga gaactg caa gtc ttg tac cgg gga ttc aaa aac gag tgc cct 304 Lys Arg Glu LeuGln Val Leu Tyr Arg Gly Phe Lys Asn Glu Cys Pro 65 70 75 agc ggt gtg gtcaat gaa gaa aca ttc aag cag atc tac gct cag ttt 352 Ser Gly Val Val AsnGlu Glu Thr Phe Lys Gln Ile Tyr Ala Gln Phe 80 85 90 ttc cct cac gga gatgcc agc aca tat gca cat tac ctc ttc aat gcc 400 Phe Pro His Gly Asp AlaSer Thr Tyr Ala His Tyr Leu Phe Asn Ala 95 100 105 ttc gac acc acc cagaca ggc tct gta aag ttc gag gac ttt gtg act 448 Phe Asp Thr Thr Gln ThrGly Ser Val Lys Phe Glu Asp Phe Val Thr 110 115 120 gct ctg tcg att ttactg aga ggg aca gtc cat gaa aaa cta agg tgg 496 Ala Leu Ser Ile Leu LeuArg Gly Thr Val His Glu Lys Leu Arg Trp 125 130 135 140 acg ttt aat ttgtat gac atc aat aaa gac ggc tac ata aac aaa gag 544 Thr Phe Asn Leu TyrAsp Ile Asn Lys Asp Gly Tyr Ile Asn Lys Glu 145 150 155 gag atg atg gacata gtc aaa gcc atc tat gac atg atg ggg aaa tac 592 Glu Met Met Asp IleVal Lys Ala Ile Tyr Asp Met Met Gly Lys Tyr 160 165 170 acc tat cct gtgctc aaa gag gac act ccc agg cag cat gtg gat gtc 640 Thr Tyr Pro Val LeuLys Glu Asp Thr Pro Arg Gln His Val Asp Val 175 180 185 ttc ttc cag aaaatg gat aaa aat aaa gat ggc att gta acg tta gat 688 Phe Phe Gln Lys MetAsp Lys Asn Lys Asp Gly Ile Val Thr Leu Asp 190 195 200 gaa ttt ctt gaatca tgt cag gag gat gac aac atc atg aga tct cta 736 Glu Phe Leu Glu SerCys Gln Glu Asp Asp Asn Ile Met Arg Ser Leu 205 210 215 220 cag ctg ttccaa aat gtc atg taactgagga cactggccat tctgctctca 787 Gln Leu Phe Gln AsnVal Met 225 gagacactga caaacacctt aatgccctga tctgcccttg ttccaattttacacaccaac 847 tcttgggaca gaaatacctt ttacactttg gaagaattct ctgctgaagactttctacaa 907 aacctggcac cacgtggctc tgtctctgag ggacgagcgg agatccgactttgttttgga 967 agcatgccca tctcttcatg ctgctgccct gtggaaggcc cctctgcttgagcttaatca 1027 atagtgcaca gttttatgct tacacatatc cccaactcac tgcctccaagtcaggcagac 1087 tctgatgaat ctgagccaaa tgtgcaccat cctccgatgg cctcccaagccaatgtgcct 1147 gcttctcttc ctctggtggg aagaaagagt gttctacgga acaattagagcttaccatga 1207 aaatattggg agaggcagca cctaacacat gtagaatagg actgaattattaagcatggt 1267 gatatcagat gatgcaaatt gcccatgtca tttttttcaa aggtagggacaaatgattct 1327 cccacactag cacctgtggt catagagcaa gtctcttaac atgcccagaaggggaaccac 1387 tgtccagtgg tctatccctc ctctccatcc cctgctcaaa cccagcactgcatgtccctc 1447 caagaaggtc cagaatgcct gcgaaacgct gtacttttat accctgttctaatcaataaa 1507 cagaactatt tcgtacaaaa aaaaaaaaaa aaa 1540 10 227 PRT Musmusculus 10 Met Gly Ala Val Met Gly Thr Phe Ser Ser Leu Gln Thr Lys GlnArg 1 5 10 15 Arg Pro Ser Lys Asp Ile Ala Trp Trp Tyr Tyr Gln Tyr GlnArg Asp 20 25 30 Lys Ile Glu Asp Glu Leu Glu Met Thr Met Val Cys His ArgPro Glu 35 40 45 Gly Leu Glu Gln Leu Glu Ala Gln Thr Asn Phe Thr Lys ArgGlu Leu 50 55 60 Gln Val Leu Tyr Arg Gly Phe Lys Asn Glu Cys Pro Ser GlyVal Val 65 70 75 80 Asn Glu Glu Thr Phe Lys Gln Ile Tyr Ala Gln Phe PhePro His Gly 85 90 95 Asp Ala Ser Thr Tyr Ala His Tyr Leu Phe Asn Ala PheAsp Thr Thr 100 105 110 Gln Thr Gly Ser Val Lys Phe Glu Asp Phe Val ThrAla Leu Ser Ile 115 120 125 Leu Leu Arg Gly Thr Val His Glu Lys Leu ArgTrp Thr Phe Asn Leu 130 135 140 Tyr Asp Ile Asn Lys Asp Gly Tyr Ile AsnLys Glu Glu Met Met Asp 145 150 155 160 Ile Val Lys Ala Ile Tyr Asp MetMet Gly Lys Tyr Thr Tyr Pro Val 165 170 175 Leu Lys Glu Asp Thr Pro ArgGln His Val Asp Val Phe Phe Gln Lys 180 185 190 Met Asp Lys Asn Lys AspGly Ile Val Thr Leu Asp Glu Phe Leu Glu 195 200 205 Ser Cys Gln Glu AspAsp Asn Ile Met Arg Ser Leu Gln Leu Phe Gln 210 215 220 Asn Val Met 22511 955 DNA Rattus sp. CDS (345)..(953) Xaa at position 92 of thecorresponding amino acid sequence may be any amino acid 11 gtccgggcacacaacccctg gattcttcgg agaatatgcc gtgacggtgt tgccaattat 60 tagttctcttggctagcaga tgtttaggga ctggttaagc ctttggagaa attaccttag 120 gaaaacggggaaataaaagc aaagattacc atgaattgca agattaccta gcaattgcaa 180 ggtaggaggagagaggtgga gggcggagta gacaggaggg agggagaaag tgagaggaag 240 ctaggctggtggaaataacc ctgcacttgg aacagcggca aagaagcgcg attttccagc 300 tttaaatgcctgcccgcgtt ctgcttgcct acccgggaac ggag atg ttg acc cag 356 Met Leu ThrGln 1 ggc gag tct gaa ggg ctc cag acc ttg ggg ata gta gtg gtc ctg tgt404 Gly Glu Ser Glu Gly Leu Gln Thr Leu Gly Ile Val Val Val Leu Cys 5 1015 20 tcc tct ctg aaa cta ctg cac tac ctc ggg ctg att gac ttg tcg gat452 Ser Ser Leu Lys Leu Leu His Tyr Leu Gly Leu Ile Asp Leu Ser Asp 2530 35 gac aag atc gag gat gat ctg gag atg acc atg gtt tgc cat cgg cct500 Asp Lys Ile Glu Asp Asp Leu Glu Met Thr Met Val Cys His Arg Pro 4045 50 gag gga ctg gag cag ctt gag gca cag acg aac ttc acc aag aga gaa548 Glu Gly Leu Glu Gln Leu Glu Ala Gln Thr Asn Phe Thr Lys Arg Glu 5560 65 ctg caa gtc ctt tac cgg gga ttc aaa aac gag tgc ccc agt ggt gtg596 Leu Gln Val Leu Tyr Arg Gly Phe Lys Asn Glu Cys Pro Ser Gly Val 7075 80 gtt aac gaa gag aca ttc aag cng atc tac gct cag ttt ttc cct cat644 Val Asn Glu Glu Thr Phe Lys Xaa Ile Tyr Ala Gln Phe Phe Pro His 8590 95 100 gga gat gcc agc aca tac gca cat tac ctc ttc aat gcc ttc gacacc 692 Gly Asp Ala Ser Thr Tyr Ala His Tyr Leu Phe Asn Ala Phe Asp Thr105 110 115 acc cag aca ggc tct gta aag ttc gag gac ttt gtg act gct ctgtcg 740 Thr Gln Thr Gly Ser Val Lys Phe Glu Asp Phe Val Thr Ala Leu Ser120 125 130 att tta ctg aga gga acg gtc cat gaa aaa ctg aag tgg acg tttaat 788 Ile Leu Leu Arg Gly Thr Val His Glu Lys Leu Lys Trp Thr Phe Asn135 140 145 ttg tac gac atc aat aaa gac ggc tac ata aac aaa gag gag atgatg 836 Leu Tyr Asp Ile Asn Lys Asp Gly Tyr Ile Asn Lys Glu Glu Met Met150 155 160 gac ata gtg aaa gcc atc tat gac atg atg ggg aaa tac acc tatctt 884 Asp Ile Val Lys Ala Ile Tyr Asp Met Met Gly Lys Tyr Thr Tyr Leu165 170 175 180 gtg ctc aaa gag gac act tcc agg cag cac gtg gac gtc ttcttc cag 932 Val Leu Lys Glu Asp Thr Ser Arg Gln His Val Asp Val Phe PheGln 185 190 195 aaa atg gat aaa aat aaa gat gg 955 Lys Met Asp Lys AsnLys Asp 200 12 203 PRT Rattus sp. 12 Met Leu Thr Gln Gly Glu Ser Glu GlyLeu Gln Thr Leu Gly Ile Val 1 5 10 15 Val Val Leu Cys Ser Ser Leu LysLeu Leu His Tyr Leu Gly Leu Ile 20 25 30 Asp Leu Ser Asp Asp Lys Ile GluAsp Asp Leu Glu Met Thr Met Val 35 40 45 Cys His Arg Pro Glu Gly Leu GluGln Leu Glu Ala Gln Thr Asn Phe 50 55 60 Thr Lys Arg Glu Leu Gln Val LeuTyr Arg Gly Phe Lys Asn Glu Cys 65 70 75 80 Pro Ser Gly Val Val Asn GluGlu Thr Phe Lys Xaa Ile Tyr Ala Gln 85 90 95 Phe Phe Pro His Gly Asp AlaSer Thr Tyr Ala His Tyr Leu Phe Asn 100 105 110 Ala Phe Asp Thr Thr GlnThr Gly Ser Val Lys Phe Glu Asp Phe Val 115 120 125 Thr Ala Leu Ser IleLeu Leu Arg Gly Thr Val His Glu Lys Leu Lys 130 135 140 Trp Thr Phe AsnLeu Tyr Asp Ile Asn Lys Asp Gly Tyr Ile Asn Lys 145 150 155 160 Glu GluMet Met Asp Ile Val Lys Ala Ile Tyr Asp Met Met Gly Lys 165 170 175 TyrThr Tyr Leu Val Leu Lys Glu Asp Thr Ser Arg Gln His Val Asp 180 185 190Val Phe Phe Gln Lys Met Asp Lys Asn Lys Asp 195 200 13 2009 DNA Homosapiens CDS (207)..(1016) 13 ctcacctgct gcctagtgtt ccctctcctg ctccaggacctccgggtaga cctcagaccc 60 cgggcccatt cccagactca gcctcagccc ggacttccccagccccgaca gcacagtagg 120 ccgccagggg gcgccgtgtg agcgccctat cccggccacccggcgccccc tcccacggcc 180 cgggcgggag cggggcgccg ggggcc atg cgg ggc cagggc cgc aag gag agt 233 Met Arg Gly Gln Gly Arg Lys Glu Ser 1 5 ttg tccgat tcc cga gac ctg gac ggc tcc tac gac cag ctc acg ggc 281 Leu Ser AspSer Arg Asp Leu Asp Gly Ser Tyr Asp Gln Leu Thr Gly 10 15 20 25 cac cctcca ggg ccc act aaa aaa gcg ctg aag cag cga ttc ctc aag 329 His Pro ProGly Pro Thr Lys Lys Ala Leu Lys Gln Arg Phe Leu Lys 30 35 40 ctg ctg ccgtgc tgc ggg ccc caa gcc ctg ccc tca gtc agt gaa aca 377 Leu Leu Pro CysCys Gly Pro Gln Ala Leu Pro Ser Val Ser Glu Thr 45 50 55 tta gcc gcc ccagcc tcc ctc cgc ccc cac aga ccc cgc ctg ctg gac 425 Leu Ala Ala Pro AlaSer Leu Arg Pro His Arg Pro Arg Leu Leu Asp 60 65 70 cca gac agc gtg gacgat gaa ttt gaa ttg tcc acc gtg tgt cac cgg 473 Pro Asp Ser Val Asp AspGlu Phe Glu Leu Ser Thr Val Cys His Arg 75 80 85 cct gag ggt ctg gag cagctg cag gag caa acc aaa ttc acg cgc aag 521 Pro Glu Gly Leu Glu Gln LeuGln Glu Gln Thr Lys Phe Thr Arg Lys 90 95 100 105 gag ttg cag gtc ctgtac cgg ggc ttc aag aac gaa tgt ccc agc gga 569 Glu Leu Gln Val Leu TyrArg Gly Phe Lys Asn Glu Cys Pro Ser Gly 110 115 120 att gtc aat gag gagaac ttc aag cag att tac tcc cag ttc ttt cct 617 Ile Val Asn Glu Glu AsnPhe Lys Gln Ile Tyr Ser Gln Phe Phe Pro 125 130 135 caa gga gac tcc agcacc tat gcc act ttt ctc ttc aat gcc ttt gac 665 Gln Gly Asp Ser Ser ThrTyr Ala Thr Phe Leu Phe Asn Ala Phe Asp 140 145 150 acc aac cat gat ggctcg gtc agt ttt gag gac ttt gtg gct ggt ttg 713 Thr Asn His Asp Gly SerVal Ser Phe Glu Asp Phe Val Ala Gly Leu 155 160 165 tcc gtg att ctt cgggga act gta gat gac agg ctt aat tgg gcc ttc 761 Ser Val Ile Leu Arg GlyThr Val Asp Asp Arg Leu Asn Trp Ala Phe 170 175 180 185 aac ctg tat gacctt aac aag gac ggc tgc atc acc aag gag gaa atg 809 Asn Leu Tyr Asp LeuAsn Lys Asp Gly Cys Ile Thr Lys Glu Glu Met 190 195 200 ctt gac atc atgaag tcc atc tat gac atg atg ggc aag tac acg tac 857 Leu Asp Ile Met LysSer Ile Tyr Asp Met Met Gly Lys Tyr Thr Tyr 205 210 215 cct gca ctc cgggag gag gcc cca agg gaa cac gtg gag agc ttc ttc 905 Pro Ala Leu Arg GluGlu Ala Pro Arg Glu His Val Glu Ser Phe Phe 220 225 230 cag aag atg gacaga aac aag gat ggt gtg gtg acc att gag gaa ttc 953 Gln Lys Met Asp ArgAsn Lys Asp Gly Val Val Thr Ile Glu Glu Phe 235 240 245 att gag tct tgtcaa aag gat gag aac atc atg agg tcc atg cag ctc 1001 Ile Glu Ser Cys GlnLys Asp Glu Asn Ile Met Arg Ser Met Gln Leu 250 255 260 265 ttt gac aatgtc atc tagcccccag gagagggggt cagtgtttcc tggggggacc 1056 Phe Asp Asn ValIle 270 atgctctaac cctagtccag gcggacctca cccttctctt cccaggtctatcctcatcct 1116 acgcctccct gggggctgga gggatccaag agcttgggga ttcagtagtccagatctctg 1176 gagctgaagg ggccagagag tgggcagagt gcatctcggg gggtgttcccaactcccacc 1236 agctctcacc cccttcctgc ctgacaccca gtgttgagag tgcccctcctgtaggaattg 1296 agcggttccc cacctcctac cctactctag aaacacacta gagcgatgtctcctgctatg 1356 gtgcttcccc catccctgac ctcataaaca tttcccctaa gactcccctctcagagagaa 1416 tgctccattc ttggcactgg ctggcttctc agaccagcca ttgagagccctgtgggaggg 1476 ggacaagaat gtatagggag aaatcttggg cctgagtcaa tggataggtcctaggaggtg 1536 ggtggggttg agaatagaag ggcctggaca gattatgatt gctcaggcataccaggttat 1596 agctccaagt tccacaggtc tgctaccaca ggccatcaaa atataagtttccaggctttg 1656 cagaagacct tgtctcctta gaaatgcccc agaaattttc cacaccctcctcggtatcca 1716 tggagagcct ggggccagat atctggctca tctctggcat tgcttcctctccttccttcc 1776 tgcatgtgtt ggtggtggtt gtggtggggg aatgtggatg ggggatgtcctggctgatgc 1836 ctgccaaaat ttcatcccac cctccttgct tatcgtccct gttttgagggctatgacttg 1896 agtttttgtt tcccatgttc tctatagact tgggaccttc ctgaacttggggcctatcac 1956 tccccacagt ggatgcctta gaagggagag ggaaggaggg aggcaggcatagc 2009 14 270 PRT Homo sapiens 14 Met Arg Gly Gln Gly Arg Lys Glu SerLeu Ser Asp Ser Arg Asp Leu 1 5 10 15 Asp Gly Ser Tyr Asp Gln Leu ThrGly His Pro Pro Gly Pro Thr Lys 20 25 30 Lys Ala Leu Lys Gln Arg Phe LeuLys Leu Leu Pro Cys Cys Gly Pro 35 40 45 Gln Ala Leu Pro Ser Val Ser GluThr Leu Ala Ala Pro Ala Ser Leu 50 55 60 Arg Pro His Arg Pro Arg Leu LeuAsp Pro Asp Ser Val Asp Asp Glu 65 70 75 80 Phe Glu Leu Ser Thr Val CysHis Arg Pro Glu Gly Leu Glu Gln Leu 85 90 95 Gln Glu Gln Thr Lys Phe ThrArg Lys Glu Leu Gln Val Leu Tyr Arg 100 105 110 Gly Phe Lys Asn Glu CysPro Ser Gly Ile Val Asn Glu Glu Asn Phe 115 120 125 Lys Gln Ile Tyr SerGln Phe Phe Pro Gln Gly Asp Ser Ser Thr Tyr 130 135 140 Ala Thr Phe LeuPhe Asn Ala Phe Asp Thr Asn His Asp Gly Ser Val 145 150 155 160 Ser PheGlu Asp Phe Val Ala Gly Leu Ser Val Ile Leu Arg Gly Thr 165 170 175 ValAsp Asp Arg Leu Asn Trp Ala Phe Asn Leu Tyr Asp Leu Asn Lys 180 185 190Asp Gly Cys Ile Thr Lys Glu Glu Met Leu Asp Ile Met Lys Ser Ile 195 200205 Tyr Asp Met Met Gly Lys Tyr Thr Tyr Pro Ala Leu Arg Glu Glu Ala 210215 220 Pro Arg Glu His Val Glu Ser Phe Phe Gln Lys Met Asp Arg Asn Lys225 230 235 240 Asp Gly Val Val Thr Ile Glu Glu Phe Ile Glu Ser Cys GlnLys Asp 245 250 255 Glu Asn Ile Met Arg Ser Met Gln Leu Phe Asp Asn ValIle 260 265 270 15 1247 DNA Rattus sp. CDS (2)..(772) 15 c cga gat ctggac ggc tcc tat gac cag ctt acg ggc cac cct cca ggg 49 Arg Asp Leu AspGly Ser Tyr Asp Gln Leu Thr Gly His Pro Pro Gly 1 5 10 15 ccc agt aaaaaa gcc ctg aag cag cgt ttc ctc aag ctg ctg ccg tgc 97 Pro Ser Lys LysAla Leu Lys Gln Arg Phe Leu Lys Leu Leu Pro Cys 20 25 30 tgc ggg ccc caagcc ctg ccc tca gtc agt gaa aca tta gct gcc cca 145 Cys Gly Pro Gln AlaLeu Pro Ser Val Ser Glu Thr Leu Ala Ala Pro 35 40 45 gcc tcc ctc cgc ccccac aga ccc cgc ccg ctg gac cca gac agc gta 193 Ala Ser Leu Arg Pro HisArg Pro Arg Pro Leu Asp Pro Asp Ser Val 50 55 60 gag gat gag ttt gaa ttatcc acg gtg tgt cac cga cct gag ggc ctg 241 Glu Asp Glu Phe Glu Leu SerThr Val Cys His Arg Pro Glu Gly Leu 65 70 75 80 gaa caa ctc cag gaa cagacc aag ttc aca cgc aga gag ctg cag gtc 289 Glu Gln Leu Gln Glu Gln ThrLys Phe Thr Arg Arg Glu Leu Gln Val 85 90 95 ctg tac cga ggc ttc aag aacgaa tgc ccc agt ggg att gtc aac gag 337 Leu Tyr Arg Gly Phe Lys Asn GluCys Pro Ser Gly Ile Val Asn Glu 100 105 110 gag aac ttc aag cag att tattct cag ttc ttt ccc caa gga gac tcc 385 Glu Asn Phe Lys Gln Ile Tyr SerGln Phe Phe Pro Gln Gly Asp Ser 115 120 125 agc aac tat gct act ttt ctcttc aat gcc ttt gac acc aac cac gat 433 Ser Asn Tyr Ala Thr Phe Leu PheAsn Ala Phe Asp Thr Asn His Asp 130 135 140 ggc tct gtc agt ttt gag gacttt gtg gct ggt ttg tcg gtg att ctt 481 Gly Ser Val Ser Phe Glu Asp PheVal Ala Gly Leu Ser Val Ile Leu 145 150 155 160 cgg ggg acc ata gat gataga ctg agc tgg gct ttc aac tta tat gac 529 Arg Gly Thr Ile Asp Asp ArgLeu Ser Trp Ala Phe Asn Leu Tyr Asp 165 170 175 ctc aac aag gac ggc tgtatc aca aag gag gaa atg ctt gac att atg 577 Leu Asn Lys Asp Gly Cys IleThr Lys Glu Glu Met Leu Asp Ile Met 180 185 190 aag tcc atc tat gac atgatg ggc aag tac aca tac cct gcc ctc cgg 625 Lys Ser Ile Tyr Asp Met MetGly Lys Tyr Thr Tyr Pro Ala Leu Arg 195 200 205 gag gag gcc cca aga gaacac gtg gag agc ttc ttc cag aag atg gac 673 Glu Glu Ala Pro Arg Glu HisVal Glu Ser Phe Phe Gln Lys Met Asp 210 215 220 agg aac aag gac ggc gtggtg acc atc gag gaa ttc atc gag tct tgt 721 Arg Asn Lys Asp Gly Val ValThr Ile Glu Glu Phe Ile Glu Ser Cys 225 230 235 240 caa cag gac gag aacatc atg agg tcc atg cag ctc ttt gat aat gtc 769 Gln Gln Asp Glu Asn IleMet Arg Ser Met Gln Leu Phe Asp Asn Val 245 250 255 atc tagctccccagggagagggg ttagtgtgtc ctagggtgac caggctgtag 822 Ile tcctagtccagacgaaccta accctctctc tccaggcctg tcctcatctt acctgtaccc 882 tgggggctgtagggattcaa tatcctgggg cttcagtagt ccagatccct gagctaagtc 942 acaaaagtaggcaagagtag gcaagctaaa tctgggggct tcccaacccc cgacagctct 1002 caccccttctcaactgatac ctagtgctga ggacacccct ggtgtaggga ccaagtggtt 1062 ctccaccttctagtcccact ctagaaacca cattagacag aaggtctcct gctatggtgc 1122 tttccccatccctaatctct tagattttcc tcaagactcc cttctcagag aacacgctct 1182 gtccatgtccccagctgggg acatggacag agcgtgttct ctagttctag atcgcgagcg 1242 gccgc 124716 257 PRT Rattus sp. 16 Arg Asp Leu Asp Gly Ser Tyr Asp Gln Leu Thr GlyHis Pro Pro Gly 1 5 10 15 Pro Ser Lys Lys Ala Leu Lys Gln Arg Phe LeuLys Leu Leu Pro Cys 20 25 30 Cys Gly Pro Gln Ala Leu Pro Ser Val Ser GluThr Leu Ala Ala Pro 35 40 45 Ala Ser Leu Arg Pro His Arg Pro Arg Pro LeuAsp Pro Asp Ser Val 50 55 60 Glu Asp Glu Phe Glu Leu Ser Thr Val Cys HisArg Pro Glu Gly Leu 65 70 75 80 Glu Gln Leu Gln Glu Gln Thr Lys Phe ThrArg Arg Glu Leu Gln Val 85 90 95 Leu Tyr Arg Gly Phe Lys Asn Glu Cys ProSer Gly Ile Val Asn Glu 100 105 110 Glu Asn Phe Lys Gln Ile Tyr Ser GlnPhe Phe Pro Gln Gly Asp Ser 115 120 125 Ser Asn Tyr Ala Thr Phe Leu PheAsn Ala Phe Asp Thr Asn His Asp 130 135 140 Gly Ser Val Ser Phe Glu AspPhe Val Ala Gly Leu Ser Val Ile Leu 145 150 155 160 Arg Gly Thr Ile AspAsp Arg Leu Ser Trp Ala Phe Asn Leu Tyr Asp 165 170 175 Leu Asn Lys AspGly Cys Ile Thr Lys Glu Glu Met Leu Asp Ile Met 180 185 190 Lys Ser IleTyr Asp Met Met Gly Lys Tyr Thr Tyr Pro Ala Leu Arg 195 200 205 Glu GluAla Pro Arg Glu His Val Glu Ser Phe Phe Gln Lys Met Asp 210 215 220 ArgAsn Lys Asp Gly Val Val Thr Ile Glu Glu Phe Ile Glu Ser Cys 225 230 235240 Gln Gln Asp Glu Asn Ile Met Arg Ser Met Gln Leu Phe Asp Asn Val 245250 255 Ile 17 2343 DNA Mus musculus CDS (181)..(990) 17 cgggactctgaggtgggccc taaaatccag cgctccccag agaaaagcct tgccagcccc 60 tactcccggcccccagcccc agcaggtcgc tgcgccgcca gggggcactg tgtgagcgcc 120 ctatcctggccacccggcgc cccctcccac ggcccaggcg ggagcggggc gccgggggcc 180 atg cgg ggccaa ggc cga aag gag agt ttg tcc gaa tcc cga gat ttg 228 Met Arg Gly GlnGly Arg Lys Glu Ser Leu Ser Glu Ser Arg Asp Leu 1 5 10 15 gac ggc tcctat gac cag ctt acg ggc cac cct cca ggg ccc agt aaa 276 Asp Gly Ser TyrAsp Gln Leu Thr Gly His Pro Pro Gly Pro Ser Lys 20 25 30 aaa gcc ctg aagcag cgt ttc ctc aag ctg ctg ccg tgc tgc ggg ccc 324 Lys Ala Leu Lys GlnArg Phe Leu Lys Leu Leu Pro Cys Cys Gly Pro 35 40 45 caa gcc ctg ccc tcagtc agt gaa aca tta gct gcc cca gcc tcc ctc 372 Gln Ala Leu Pro Ser ValSer Glu Thr Leu Ala Ala Pro Ala Ser Leu 50 55 60 cgc ccc cac aga ccc cgcccg ctg gac cca gac agc gtg gag gat gag 420 Arg Pro His Arg Pro Arg ProLeu Asp Pro Asp Ser Val Glu Asp Glu 65 70 75 80 ttt gaa cta tcc acg gtgtgc cac cgg cct gag ggt ctg gaa caa ctc 468 Phe Glu Leu Ser Thr Val CysHis Arg Pro Glu Gly Leu Glu Gln Leu 85 90 95 cag gaa caa acc aag ttc acacgc aga gag ttg cag gtc ctg tac aga 516 Gln Glu Gln Thr Lys Phe Thr ArgArg Glu Leu Gln Val Leu Tyr Arg 100 105 110 ggc ttc aag aac gaa tgt cccagc gga att gtc aac gag gag aac ttc 564 Gly Phe Lys Asn Glu Cys Pro SerGly Ile Val Asn Glu Glu Asn Phe 115 120 125 aag caa att tat tct cag ttcttt ccc caa gga gac tcc agc aac tac 612 Lys Gln Ile Tyr Ser Gln Phe PhePro Gln Gly Asp Ser Ser Asn Tyr 130 135 140 gct act ttt ctc ttc aat gccttt gac acc aac cat gat ggc tct gtc 660 Ala Thr Phe Leu Phe Asn Ala PheAsp Thr Asn His Asp Gly Ser Val 145 150 155 160 agt ttt gag gac ttt gtggct ggt ttg tca gtg att ctt cgg gga acc 708 Ser Phe Glu Asp Phe Val AlaGly Leu Ser Val Ile Leu Arg Gly Thr 165 170 175 ata gat gat aga ctg aactgg gct ttc aac tta tat gac ctc aac aag 756 Ile Asp Asp Arg Leu Asn TrpAla Phe Asn Leu Tyr Asp Leu Asn Lys 180 185 190 gat ggc tgt atc acg aaggag gaa atg ctc gac atc atg aag tcc atc 804 Asp Gly Cys Ile Thr Lys GluGlu Met Leu Asp Ile Met Lys Ser Ile 195 200 205 tat gac atg atg ggc aagtac acc tac cct gcc ctc cgg gag gag gcc 852 Tyr Asp Met Met Gly Lys TyrThr Tyr Pro Ala Leu Arg Glu Glu Ala 210 215 220 ccg agg gaa cac gtg gagagc ttc ttc cag aag atg gac aga aac aag 900 Pro Arg Glu His Val Glu SerPhe Phe Gln Lys Met Asp Arg Asn Lys 225 230 235 240 gac ggc gtg gtg accatt gag gaa ttc att gag tct tgt caa cag gac 948 Asp Gly Val Val Thr IleGlu Glu Phe Ile Glu Ser Cys Gln Gln Asp 245 250 255 gag aac atc atg aggtcc atg caa ctc ttt gat aat gtc atc 990 Glu Asn Ile Met Arg Ser Met GlnLeu Phe Asp Asn Val Ile 260 265 270 tagctcccca gggagagggg ttagtgtgtcccagggtaac catgctgtag ccctagtcca 1050 ggcaaaccta accctcctct ccccgggtctgtcctcatcc tacctgtacc ctgggggctg 1110 tagggattca acatcctggc gcttcagtagtccagatccc tgagctaagt ggcgagagta 1170 ggcaagctaa gtctttggag ggtgggtgggggcgcgcaga ttcccaaccc ccgacgactc 1230 tcaccccttt ctcgactgat acccagtgctgaggctaccc ctggtgtcgg gaacgaccaa 1290 agtggttctc tgcctcccca gcccactctagagacccaca ctagacggga atatctcctg 1350 ctatggtgct ttccccatcc ctgaccgcagattttcctcc taagactccc ttctcagaga 1410 atatgctttt gtcccttgtc cctggctggcttttcagcct agcctttgag gaccctgtgg 1470 gaggggagaa taagaaagca gacaaaatcttggccctgag ccagtggtta ggtcctagga 1530 atcaggctgg agtggagacc agaaagcctgggcaggctat gagagcccca ggttggcttg 1590 tcaccgccag gttccacagg gctgctgctctgggtcagca gagtatgagt ttccagactt 1650 tccagaaggc cttatgtcct tagcaatgtcccagaaattc accatacact tctcagtgtc 1710 ttaggatcca gatgtccggt ccatccctgaaacctctccc tcctccttgc tcctatggtg 1770 ggagtggtgg ccaggggacg atgagtgagccggtgtcctg gatgatgcct gtcaaggtcc 1830 cacctaccct ccggctgtca agccgttctggtgaccctgt ttgattctcc atgacccctg 1890 tctagatgta gaggtgtgga gtgagtctagtggcagcctt aggggaatgg gaagaacgag 1950 aggggcactc catctgaacc cagtgtgggggcatccattc gaatctttgc ctggctcccc 2010 acaatgccct aggatcctct agggtccccacccccactct ttagtctacc cagagatgct 2070 ccagagctca cctagagggc agggaccataggatccaggt ccaacctgtc atcagcatcc 2130 ggccatgctg ctgctgctta ttaataaacctgcttgtcgt tcagcgcccc ttcccagtca 2190 gccagggtct gaggggaagg cccccactttcccgcctcct gtcagacatt gttgactgct 2250 ttgcattttg ggctcttcta cctatattttgtataataag aaagacacca gatccaataa 2310 aacacatggc tatgcacaaa aaaaaaaaaaaaa 2343 18 270 PRT Mus musculus 18 Met Arg Gly Gln Gly Arg Lys Glu SerLeu Ser Glu Ser Arg Asp Leu 1 5 10 15 Asp Gly Ser Tyr Asp Gln Leu ThrGly His Pro Pro Gly Pro Ser Lys 20 25 30 Lys Ala Leu Lys Gln Arg Phe LeuLys Leu Leu Pro Cys Cys Gly Pro 35 40 45 Gln Ala Leu Pro Ser Val Ser GluThr Leu Ala Ala Pro Ala Ser Leu 50 55 60 Arg Pro His Arg Pro Arg Pro LeuAsp Pro Asp Ser Val Glu Asp Glu 65 70 75 80 Phe Glu Leu Ser Thr Val CysHis Arg Pro Glu Gly Leu Glu Gln Leu 85 90 95 Gln Glu Gln Thr Lys Phe ThrArg Arg Glu Leu Gln Val Leu Tyr Arg 100 105 110 Gly Phe Lys Asn Glu CysPro Ser Gly Ile Val Asn Glu Glu Asn Phe 115 120 125 Lys Gln Ile Tyr SerGln Phe Phe Pro Gln Gly Asp Ser Ser Asn Tyr 130 135 140 Ala Thr Phe LeuPhe Asn Ala Phe Asp Thr Asn His Asp Gly Ser Val 145 150 155 160 Ser PheGlu Asp Phe Val Ala Gly Leu Ser Val Ile Leu Arg Gly Thr 165 170 175 IleAsp Asp Arg Leu Asn Trp Ala Phe Asn Leu Tyr Asp Leu Asn Lys 180 185 190Asp Gly Cys Ile Thr Lys Glu Glu Met Leu Asp Ile Met Lys Ser Ile 195 200205 Tyr Asp Met Met Gly Lys Tyr Thr Tyr Pro Ala Leu Arg Glu Glu Ala 210215 220 Pro Arg Glu His Val Glu Ser Phe Phe Gln Lys Met Asp Arg Asn Lys225 230 235 240 Asp Gly Val Val Thr Ile Glu Glu Phe Ile Glu Ser Cys GlnGln Asp 245 250 255 Glu Asn Ile Met Arg Ser Met Gln Leu Phe Asp Asn ValIle 260 265 270 19 1955 DNA Homo sapiens CDS (207)..(962) 19 ctcacctgctgcctagtgtt ccctctcctg ctccaggacc tccgggtaga cctcagaccc 60 cgggcccattcccagactca gcctcagccc ggacttcccc agccccgaca gcacagtagg 120 ccgccagggggcgccgtgtg agcgccctat cccggccacc cggcgccccc tcccacggcc 180 cgggcgggagcggggcgccg ggggcc atg cgg ggc cag ggc cgc aag gag agt 233 Met Arg GlyGln Gly Arg Lys Glu Ser 1 5 ttg tcc gat tcc cga gac ctg gac ggc tcc tacgac cag ctc acg ggc 281 Leu Ser Asp Ser Arg Asp Leu Asp Gly Ser Tyr AspGln Leu Thr Gly 10 15 20 25 cac cct cca ggg ccc act aaa aaa gcg ctg aagcag cga ttc ctc aag 329 His Pro Pro Gly Pro Thr Lys Lys Ala Leu Lys GlnArg Phe Leu Lys 30 35 40 ctg ctg ccg tgc tgc ggg ccc caa gcc ctg ccc tcagtc agt gaa aac 377 Leu Leu Pro Cys Cys Gly Pro Gln Ala Leu Pro Ser ValSer Glu Asn 45 50 55 agc gtg gac gat gaa ttt gaa ttg tcc acc gtg tgt caccgg cct gag 425 Ser Val Asp Asp Glu Phe Glu Leu Ser Thr Val Cys His ArgPro Glu 60 65 70 ggt ctg gag cag ctg cag gag caa acc aaa ttc acg cgc aaggag ttg 473 Gly Leu Glu Gln Leu Gln Glu Gln Thr Lys Phe Thr Arg Lys GluLeu 75 80 85 cag gtc ctg tac cgg ggc ttc aag aac gaa tgt ccc agc gga attgtc 521 Gln Val Leu Tyr Arg Gly Phe Lys Asn Glu Cys Pro Ser Gly Ile Val90 95 100 105 aat gag gag aac ttc aag cag att tac tcc cag ttc ttt cctcaa gga 569 Asn Glu Glu Asn Phe Lys Gln Ile Tyr Ser Gln Phe Phe Pro GlnGly 110 115 120 gac tcc agc acc tat gcc act ttt ctc ttc aat gcc ttt gacacc aac 617 Asp Ser Ser Thr Tyr Ala Thr Phe Leu Phe Asn Ala Phe Asp ThrAsn 125 130 135 cat gat ggc tcg gtc agt ttt gag gac ttt gtg gct ggt ttgtcc gtg 665 His Asp Gly Ser Val Ser Phe Glu Asp Phe Val Ala Gly Leu SerVal 140 145 150 att ctt cgg gga act gta gat gac agg ctt aat tgg gcc ttcaac ctg 713 Ile Leu Arg Gly Thr Val Asp Asp Arg Leu Asn Trp Ala Phe AsnLeu 155 160 165 tat gac ctt aac aag gac ggc tgc atc acc aag gag gaa atgctt gac 761 Tyr Asp Leu Asn Lys Asp Gly Cys Ile Thr Lys Glu Glu Met LeuAsp 170 175 180 185 atc atg aag tcc atc tat gac atg atg ggc aag tac acgtac cct gca 809 Ile Met Lys Ser Ile Tyr Asp Met Met Gly Lys Tyr Thr TyrPro Ala 190 195 200 ctc cgg gag gag gcc cca agg gaa cac gtg gag agc ttcttc cag aag 857 Leu Arg Glu Glu Ala Pro Arg Glu His Val Glu Ser Phe PheGln Lys 205 210 215 atg gac aga aac aag gat ggt gtg gtg acc att gag gaattc att gag 905 Met Asp Arg Asn Lys Asp Gly Val Val Thr Ile Glu Glu PheIle Glu 220 225 230 tct tgt caa aag gat gag aac atc atg agg tcc atg cagctc ttt gac 953 Ser Cys Gln Lys Asp Glu Asn Ile Met Arg Ser Met Gln LeuPhe Asp 235 240 245 aat gtc atc tagcccccag gagagggggt cagtgtttcctggggggacc 1002 Asn Val Ile 250 atgctctaac cctagtccag gcggacctcacccttctctt cccaggtcta tcctcatcct 1062 acgcctccct gggggctgga gggatccaagagcttgggga ttcagtagtc cagatctctg 1122 gagctgaagg ggccagagag tgggcagagtgcatctcggg gggtgttccc aactcccacc 1182 agctctcacc cccttcctgc ctgacacccagtgttgagag tgcccctcct gtaggaattg 1242 agcggttccc cacctcctac cctactctagaaacacacta gagcgatgtc tcctgctatg 1302 gtgcttcccc catccctgac ctcataaacatttcccctaa gactcccctc tcagagagaa 1362 tgctccattc ttggcactgg ctggcttctcagaccagcca ttgagagccc tgtgggaggg 1422 ggacaagaat gtatagggag aaatcttgggcctgagtcaa tggataggtc ctaggaggtg 1482 ggtggggttg agaatagaag ggcctggacagattatgatt gctcaggcat accaggttat 1542 agctccaagt tccacaggtc tgctaccacaggccatcaaa atataagttt ccaggctttg 1602 cagaagacct tgtctcctta gaaatgccccagaaattttc cacaccctcc tcggtatcca 1662 tggagagcct ggggccagat atctggctcatctctggcat tgcttcctct ccttccttcc 1722 tgcatgtgtt ggtggtggtt gtggtgggggaatgtggatg ggggatgtcc tggctgatgc 1782 ctgccaaaat ttcatcccac cctccttgcttatcgtccct gttttgaggg ctatgacttg 1842 agtttttgtt tcccatgttc tctatagacttgggaccttc ctgaacttgg ggcctatcac 1902 tccccacagt ggatgcctta gaagggagagggaaggaggg aggcaggcat agc 1955 20 252 PRT Homo sapiens 20 Met Arg GlyGln Gly Arg Lys Glu Ser Leu Ser Asp Ser Arg Asp Leu 1 5 10 15 Asp GlySer Tyr Asp Gln Leu Thr Gly His Pro Pro Gly Pro Thr Lys 20 25 30 Lys AlaLeu Lys Gln Arg Phe Leu Lys Leu Leu Pro Cys Cys Gly Pro 35 40 45 Gln AlaLeu Pro Ser Val Ser Glu Asn Ser Val Asp Asp Glu Phe Glu 50 55 60 Leu SerThr Val Cys His Arg Pro Glu Gly Leu Glu Gln Leu Gln Glu 65 70 75 80 GlnThr Lys Phe Thr Arg Lys Glu Leu Gln Val Leu Tyr Arg Gly Phe 85 90 95 LysAsn Glu Cys Pro Ser Gly Ile Val Asn Glu Glu Asn Phe Lys Gln 100 105 110Ile Tyr Ser Gln Phe Phe Pro Gln Gly Asp Ser Ser Thr Tyr Ala Thr 115 120125 Phe Leu Phe Asn Ala Phe Asp Thr Asn His Asp Gly Ser Val Ser Phe 130135 140 Glu Asp Phe Val Ala Gly Leu Ser Val Ile Leu Arg Gly Thr Val Asp145 150 155 160 Asp Arg Leu Asn Trp Ala Phe Asn Leu Tyr Asp Leu Asn LysAsp Gly 165 170 175 Cys Ile Thr Lys Glu Glu Met Leu Asp Ile Met Lys SerIle Tyr Asp 180 185 190 Met Met Gly Lys Tyr Thr Tyr Pro Ala Leu Arg GluGlu Ala Pro Arg 195 200 205 Glu His Val Glu Ser Phe Phe Gln Lys Met AspArg Asn Lys Asp Gly 210 215 220 Val Val Thr Ile Glu Glu Phe Ile Glu SerCys Gln Lys Asp Glu Asn 225 230 235 240 Ile Met Arg Ser Met Gln Leu PheAsp Asn Val Ile 245 250 21 2300 DNA Rattus sp. CDS (214)..(969) 21ctcacttgct gcccaaggct cctgctcctg ccccaggact ctgaggtggg ccctaaaacc 60cagcgctctc taaagaaaag ccttgccagc ccctactccc ggcccccaac cccagcaggt 120cgctgcgccg ccagggggcg ctgtgtgagc gccctattct ggccacccgg cgccccctcc 180cacggcccag gcgggagcgg ggcgccgggg gcc atg cgg ggc caa ggc aga aag 234 MetArg Gly Gln Gly Arg Lys 1 5 gag agt ttg tcc gaa tcc cga gat ctg gac ggctcc tat gac cag ctt 282 Glu Ser Leu Ser Glu Ser Arg Asp Leu Asp Gly SerTyr Asp Gln Leu 10 15 20 acg ggc cac cct cca ggg ccc agt aaa aaa gcc ctgaag cag cgt ttc 330 Thr Gly His Pro Pro Gly Pro Ser Lys Lys Ala Leu LysGln Arg Phe 25 30 35 ctc aag ctg ctg ccg tgc tgc ggg ccc caa gcc ctg ccctca gtc agt 378 Leu Lys Leu Leu Pro Cys Cys Gly Pro Gln Ala Leu Pro SerVal Ser 40 45 50 55 gaa aac agc gta gag gat gag ttt gaa tta tcc acg gtgtgt cac cga 426 Glu Asn Ser Val Glu Asp Glu Phe Glu Leu Ser Thr Val CysHis Arg 60 65 70 cct gag ggc ctg gaa caa ctc cag gaa cag acc aag ttc acacgc aga 474 Pro Glu Gly Leu Glu Gln Leu Gln Glu Gln Thr Lys Phe Thr ArgArg 75 80 85 gag ctg cag gtc ctg tac cga ggc ttc aag aac gaa tgc ccc agtggg 522 Glu Leu Gln Val Leu Tyr Arg Gly Phe Lys Asn Glu Cys Pro Ser Gly90 95 100 att gtc aac gag gag aac ttc aag cag att tat tct cag ttc tttccc 570 Ile Val Asn Glu Glu Asn Phe Lys Gln Ile Tyr Ser Gln Phe Phe Pro105 110 115 caa gga gac tcc agc aac tat gct act ttt ctc ttc aat gcc tttgac 618 Gln Gly Asp Ser Ser Asn Tyr Ala Thr Phe Leu Phe Asn Ala Phe Asp120 125 130 135 acc aac cac gat ggc tct gtc agt ttt gag gac ttt gtg gctggt ttg 666 Thr Asn His Asp Gly Ser Val Ser Phe Glu Asp Phe Val Ala GlyLeu 140 145 150 tcg gtg att ctt cgg ggg acc ata gat gat aga ctg agc tgggct ttc 714 Ser Val Ile Leu Arg Gly Thr Ile Asp Asp Arg Leu Ser Trp AlaPhe 155 160 165 aac tta tat gac ctc aac aag gac ggc tgt atc aca aag gaggaa atg 762 Asn Leu Tyr Asp Leu Asn Lys Asp Gly Cys Ile Thr Lys Glu GluMet 170 175 180 ctt gac att atg aag tcc atc tat gac atg atg ggc aag tacaca tac 810 Leu Asp Ile Met Lys Ser Ile Tyr Asp Met Met Gly Lys Tyr ThrTyr 185 190 195 cct gcc ctc cgg gag gag gcc cca aga gaa cac gtg gag agcttc ttc 858 Pro Ala Leu Arg Glu Glu Ala Pro Arg Glu His Val Glu Ser PhePhe 200 205 210 215 cag aag atg gac agg aac aag gac ggc gtg gtg acc atcgag gaa ttc 906 Gln Lys Met Asp Arg Asn Lys Asp Gly Val Val Thr Ile GluGlu Phe 220 225 230 atc gag tct tgt caa cag gac gag aac atc atg agg tccatg cag ctc 954 Ile Glu Ser Cys Gln Gln Asp Glu Asn Ile Met Arg Ser MetGln Leu 235 240 245 ttt gat aat gtc atc tagctcccca gggagagggg ttagtgtgtcctagggtgac 1009 Phe Asp Asn Val Ile 250 caggctgtag tcctagtcca gacgaacctaaccctctctc tccaggcctg tcctcatctt 1069 acctgtaccc tgggggctgt agggattcaatatcctgggg cttcagtagt ccagatccct 1129 gagctaagtc acaaaagtag gcaagagtaggcaagctaaa tctgggggct tcccaacccc 1189 cgacagctct caccccttct caactgatacctagtgctga ggacacccct ggtgtaggga 1249 ccaagtggtt ctccaccttc tagtcccactctagaaacca cattagacag aaggtctcct 1309 gctatggtgc tttccccatc cctaatctcttagattttcc tcaagactcc cttctcagag 1369 aacacgctct gtccatgtcc ccagctggcttctcagccta gcctttgagg gccctgtggg 1429 gaggcgggga caagaaagca gaaaagtcttggccccgagc cagtggttag gtcctaggaa 1489 ttggctggag tggaggccag aaagcctgggcagatgatga gagcccagct gggctgtcac 1549 tgcaggttcc ggggcctaca gccctgggtcagcagagtat gagttcccag actttccaga 1609 aggtccttag caatgtccca gaaattcaccgtacacttct cagtgtctta ggagggcccg 1669 ggatccagat gtctggttca tccctgaatcctctccctcc ttcttgctcg tatggtggga 1729 gtggtggcca ggggaagatg agtggtgtcccggatgatgc ctgtcaaggt cccacctccc 1789 ctccggctgt tctcatgaca gctgtttggttctccatgac ccctatctag atgtagaggc 1849 atggagtgag tcagggattt cccgaacttgagttttacca ctcctcctag tggctgcctt 1909 aggggaatgg gaagaaccca gtgtgggggcacccattaga atctttgccc ggctcctcac 1969 aatgccctag ggtcccctag ggtacccgctccctctgttt agtctaccca gagatgctcc 2029 tgagctcacc tagagggtag ggacggtaggctccaggtcc aacctctcca ggtcagcacc 2089 ctgccatgct gctgctcctc attaacaaacctgcttgtct cctcctgcgc cccttctcag 2149 tcagccaggg tctgagggga agggcctcccgtttccccat ccgtcagaca tggttgactg 2209 ctttgcattt tgggctcttc tatctattttgtaaaataag acatcagatc caataaaaca 2269 cacggctatg cacaaaaaaa aaaaaaaaaa a2300 22 252 PRT Rattus sp. 22 Met Arg Gly Gln Gly Arg Lys Glu Ser LeuSer Glu Ser Arg Asp Leu 1 5 10 15 Asp Gly Ser Tyr Asp Gln Leu Thr GlyHis Pro Pro Gly Pro Ser Lys 20 25 30 Lys Ala Leu Lys Gln Arg Phe Leu LysLeu Leu Pro Cys Cys Gly Pro 35 40 45 Gln Ala Leu Pro Ser Val Ser Glu AsnSer Val Glu Asp Glu Phe Glu 50 55 60 Leu Ser Thr Val Cys His Arg Pro GluGly Leu Glu Gln Leu Gln Glu 65 70 75 80 Gln Thr Lys Phe Thr Arg Arg GluLeu Gln Val Leu Tyr Arg Gly Phe 85 90 95 Lys Asn Glu Cys Pro Ser Gly IleVal Asn Glu Glu Asn Phe Lys Gln 100 105 110 Ile Tyr Ser Gln Phe Phe ProGln Gly Asp Ser Ser Asn Tyr Ala Thr 115 120 125 Phe Leu Phe Asn Ala PheAsp Thr Asn His Asp Gly Ser Val Ser Phe 130 135 140 Glu Asp Phe Val AlaGly Leu Ser Val Ile Leu Arg Gly Thr Ile Asp 145 150 155 160 Asp Arg LeuSer Trp Ala Phe Asn Leu Tyr Asp Leu Asn Lys Asp Gly 165 170 175 Cys IleThr Lys Glu Glu Met Leu Asp Ile Met Lys Ser Ile Tyr Asp 180 185 190 MetMet Gly Lys Tyr Thr Tyr Pro Ala Leu Arg Glu Glu Ala Pro Arg 195 200 205Glu His Val Glu Ser Phe Phe Gln Lys Met Asp Arg Asn Lys Asp Gly 210 215220 Val Val Thr Ile Glu Glu Phe Ile Glu Ser Cys Gln Gln Asp Glu Asn 225230 235 240 Ile Met Arg Ser Met Gln Leu Phe Asp Asn Val Ile 245 250 231859 DNA Homo sapiens CDS (207)..(866) 23 ctcacctgct gcctagtgttccctctcctg ctccaggacc tccgggtaga cctcagaccc 60 cgggcccatt cccagactcagcctcagccc ggacttcccc agccccgaca gcacagtagg 120 ccgccagggg gcgccgtgtgagcgccctat cccggccacc cggcgccccc tcccacggcc 180 cgggcgggag cggggcgccgggggcc atg cgg ggc cag ggc cgc aag gag agt 233 Met Arg Gly Gln Gly ArgLys Glu Ser 1 5 ttg tcc gat tcc cga gac ctg gac ggc tcc tac gac cag ctcacg gac 281 Leu Ser Asp Ser Arg Asp Leu Asp Gly Ser Tyr Asp Gln Leu ThrAsp 10 15 20 25 agc gtg gac gat gaa ttt gaa ttg tcc acc gtg tgt cac cggcct gag 329 Ser Val Asp Asp Glu Phe Glu Leu Ser Thr Val Cys His Arg ProGlu 30 35 40 ggt ctg gag cag ctg cag gag caa acc aaa ttc acg cgc aag gagttg 377 Gly Leu Glu Gln Leu Gln Glu Gln Thr Lys Phe Thr Arg Lys Glu Leu45 50 55 cag gtc ctg tac cgg ggc ttc aag aac gaa tgt ccc agc gga att gtc425 Gln Val Leu Tyr Arg Gly Phe Lys Asn Glu Cys Pro Ser Gly Ile Val 6065 70 aat gag gag aac ttc aag cag att tac tcc cag ttc ttt cct caa gga473 Asn Glu Glu Asn Phe Lys Gln Ile Tyr Ser Gln Phe Phe Pro Gln Gly 7580 85 gac tcc agc acc tat gcc act ttt ctc ttc aat gcc ttt gac acc aac521 Asp Ser Ser Thr Tyr Ala Thr Phe Leu Phe Asn Ala Phe Asp Thr Asn 9095 100 105 cat gat ggc tcg gtc agt ttt gag gac ttt gtg gct ggt ttg tccgtg 569 His Asp Gly Ser Val Ser Phe Glu Asp Phe Val Ala Gly Leu Ser Val110 115 120 att ctt cgg gga act gta gat gac agg ctt aat tgg gcc ttc aacctg 617 Ile Leu Arg Gly Thr Val Asp Asp Arg Leu Asn Trp Ala Phe Asn Leu125 130 135 tat gac ctt aac aag gac ggc tgc atc acc aag gag gaa atg cttgac 665 Tyr Asp Leu Asn Lys Asp Gly Cys Ile Thr Lys Glu Glu Met Leu Asp140 145 150 atc atg aag tcc atc tat gac atg atg ggc aag tac acg tac cctgca 713 Ile Met Lys Ser Ile Tyr Asp Met Met Gly Lys Tyr Thr Tyr Pro Ala155 160 165 ctc cgg gag gag gcc cca agg gaa cac gtg gag agc ttc ttc cagaag 761 Leu Arg Glu Glu Ala Pro Arg Glu His Val Glu Ser Phe Phe Gln Lys170 175 180 185 atg gac aga aac aag gat ggt gtg gtg acc att gag gaa ttcatt gag 809 Met Asp Arg Asn Lys Asp Gly Val Val Thr Ile Glu Glu Phe IleGlu 190 195 200 tct tgt caa aag gat gag aac atc atg agg tcc atg cag ctcttt gac 857 Ser Cys Gln Lys Asp Glu Asn Ile Met Arg Ser Met Gln Leu PheAsp 205 210 215 aat gtc atc tagcccccag gagagggggt cagtgtttcc tggggggacc906 Asn Val Ile 220 atgctctaac cctagtccag gcggacctca cccttctcttcccaggtcta tcctcatcct 966 acgcctccct gggggctgga gggatccaag agcttggggattcagtagtc cagatctctg 1026 gagctgaagg ggccagagag tgggcagagt gcatctcggggggtgttccc aactcccacc 1086 agctctcacc cccttcctgc ctgacaccca gtgttgagagtgcccctcct gtaggaattg 1146 agcggttccc cacctcctac cctactctag aaacacactagagcgatgtc tcctgctatg 1206 gtgcttcccc catccctgac ctcataaaca tttcccctaagactcccctc tcagagagaa 1266 tgctccattc ttggcactgg ctggcttctc agaccagccattgagagccc tgtgggaggg 1326 ggacaagaat gtatagggag aaatcttggg cctgagtcaatggataggtc ctaggaggtg 1386 ggtggggttg agaatagaag ggcctggaca gattatgattgctcaggcat accaggttat 1446 agctccaagt tccacaggtc tgctaccaca ggccatcaaaatataagttt ccaggctttg 1506 cagaagacct tgtctcctta gaaatgcccc agaaattttccacaccctcc tcggtatcca 1566 tggagagcct ggggccagat atctggctca tctctggcattgcttcctct ccttccttcc 1626 tgcatgtgtt ggtggtggtt gtggtggggg aatgtggatgggggatgtcc tggctgatgc 1686 ctgccaaaat ttcatcccac cctccttgct tatcgtccctgttttgaggg ctatgacttg 1746 agtttttgtt tcccatgttc tctatagact tgggaccttcctgaacttgg ggcctatcac 1806 tccccacagt ggatgcctta gaagggagag ggaaggagggaggcaggcat agc 1859 24 220 PRT Homo sapiens 24 Met Arg Gly Gln Gly ArgLys Glu Ser Leu Ser Asp Ser Arg Asp Leu 1 5 10 15 Asp Gly Ser Tyr AspGln Leu Thr Asp Ser Val Asp Asp Glu Phe Glu 20 25 30 Leu Ser Thr Val CysHis Arg Pro Glu Gly Leu Glu Gln Leu Gln Glu 35 40 45 Gln Thr Lys Phe ThrArg Lys Glu Leu Gln Val Leu Tyr Arg Gly Phe 50 55 60 Lys Asn Glu Cys ProSer Gly Ile Val Asn Glu Glu Asn Phe Lys Gln 65 70 75 80 Ile Tyr Ser GlnPhe Phe Pro Gln Gly Asp Ser Ser Thr Tyr Ala Thr 85 90 95 Phe Leu Phe AsnAla Phe Asp Thr Asn His Asp Gly Ser Val Ser Phe 100 105 110 Glu Asp PheVal Ala Gly Leu Ser Val Ile Leu Arg Gly Thr Val Asp 115 120 125 Asp ArgLeu Asn Trp Ala Phe Asn Leu Tyr Asp Leu Asn Lys Asp Gly 130 135 140 CysIle Thr Lys Glu Glu Met Leu Asp Ile Met Lys Ser Ile Tyr Asp 145 150 155160 Met Met Gly Lys Tyr Thr Tyr Pro Ala Leu Arg Glu Glu Ala Pro Arg 165170 175 Glu His Val Glu Ser Phe Phe Gln Lys Met Asp Arg Asn Lys Asp Gly180 185 190 Val Val Thr Ile Glu Glu Phe Ile Glu Ser Cys Gln Lys Asp GluAsn 195 200 205 Ile Met Arg Ser Met Gln Leu Phe Asp Asn Val Ile 210 215220 25 2191 DNA Simian sp. CDS (133)..(792) 25 cccacgcgtc cgcccacgcgtccgcggacg cgtggggtgc actaggccgc cagggggcgc 60 cgtgtgagcg ccctatcccggccacccggc gccccctccc acggaccggg cgggagcggg 120 gcgccggggg cc atg cggggc cag ggc cgc aag gag agt ttg tcc gat tcc 171 Met Arg Gly Gln Gly ArgLys Glu Ser Leu Ser Asp Ser 1 5 10 cga gac ctg gac gga tcc tac gac cagctc acg gac agc gtg gag gat 219 Arg Asp Leu Asp Gly Ser Tyr Asp Gln LeuThr Asp Ser Val Glu Asp 15 20 25 gaa ttt gaa ttg tcc acc gtg tgt cac cggcct gag ggt ctg gag cag 267 Glu Phe Glu Leu Ser Thr Val Cys His Arg ProGlu Gly Leu Glu Gln 30 35 40 45 ctg cag gag caa acc aaa ttc acg cgc aaggag ttg cag gtc ctg tac 315 Leu Gln Glu Gln Thr Lys Phe Thr Arg Lys GluLeu Gln Val Leu Tyr 50 55 60 cgg ggc ttc aag aac gaa tgt ccg agc gga attgtc aat gag gag aac 363 Arg Gly Phe Lys Asn Glu Cys Pro Ser Gly Ile ValAsn Glu Glu Asn 65 70 75 ttc aag caa att tac tcc cag ttc ttt cct caa ggagac tcc agc acc 411 Phe Lys Gln Ile Tyr Ser Gln Phe Phe Pro Gln Gly AspSer Ser Thr 80 85 90 tat gcc act ttt ctc ttc aat gcc ttt gac acc aac catgat ggc tcg 459 Tyr Ala Thr Phe Leu Phe Asn Ala Phe Asp Thr Asn His AspGly Ser 95 100 105 gtc agt ttt gag gac ttt gtg gct ggt ttg tcc gtg attctt cgg gga 507 Val Ser Phe Glu Asp Phe Val Ala Gly Leu Ser Val Ile LeuArg Gly 110 115 120 125 act gta gat gac agg ctt aat tgg gcc ttc aac ttgtat gac ctc aac 555 Thr Val Asp Asp Arg Leu Asn Trp Ala Phe Asn Leu TyrAsp Leu Asn 130 135 140 aag gac ggc tgc atc acc aag gag gaa atg ctt gacatc atg aag tcc 603 Lys Asp Gly Cys Ile Thr Lys Glu Glu Met Leu Asp IleMet Lys Ser 145 150 155 atc tat gac atg atg ggc aag tac aca tac cct gcactc cgg gag gag 651 Ile Tyr Asp Met Met Gly Lys Tyr Thr Tyr Pro Ala LeuArg Glu Glu 160 165 170 gcc cca agg gaa cat gtg gag aac ttc ttc cag aagatg gac aga aac 699 Ala Pro Arg Glu His Val Glu Asn Phe Phe Gln Lys MetAsp Arg Asn 175 180 185 aag gat ggc gtg gtg acc att gag gaa ttc att gagtct tgt caa aag 747 Lys Asp Gly Val Val Thr Ile Glu Glu Phe Ile Glu SerCys Gln Lys 190 195 200 205 gat gag aac atc atg agg tcc atg cag ctc tttgac aat gtc atc 792 Asp Glu Asn Ile Met Arg Ser Met Gln Leu Phe Asp AsnVal Ile 210 215 220 tagcccccag gagagggggt cagtgtttcc tggggggaccatgctctaac cctagtccag 852 gtggacctca cccttctctt cccaggtcta tccttgtcctaggcctccct gggggctgga 912 gggatccaag agcttgggga ttcagtagtc cagatctctggagctgaagg ggccagagag 972 tgggcagagt gcatcttggg gggtgttccc aactcccaccagctttcacc cgcttcctgc 1032 ctgacaccca gtgttgagag tgcccctcct gtaggaactgagtggttccc cacctcctac 1092 ccccactcta gaaacacact agacagatgt ctcctgctatggtgcttccc ccatccctga 1152 cttcataaac atttccccta aaactccctt ctcagagagaatgctccatt cttggcactg 1212 gctggcttct cagaccagcc tttgagagcc ctgtgggagggggacaagaa tgtatagggg 1272 agaaatcttg ggcctgagtc aatggatagg tcctaggaggtggctggggt tgagaataga 1332 aaggcctgga cacaatgtga ttgctcaggc ataccaagttatagctccaa gttccacagg 1392 tctgctacca caggccatca aaatataagt ttccaggctttgcagaagac cttgtctcct 1452 tggaaatgcc ccagatattt tccataccct cctcgatatccatggagagc ctggggctag 1512 atatctggca tatccctggc attgcttcct ctccttccttcctgcatgtg ttggtggtgg 1572 ttgtggcagg ggaatgtgga taggagatgt cctggcagatgcctgccaaa gtttcatccc 1632 accctccctg ctcatcgccc ctgttttgag ggctgtgacttgagtttttg tttcccatgt 1692 tctctataga cttgggacct tcctgaactt ggggcctatcactccccaca gtggatgcct 1752 tagaagggag agggaaggag ggaggcaggc atagcatctgaacccagtgt gggggcattc 1812 actaggatct tcaatcaacc cgggctctcc ccaaccccccagataacctc ctcagttccc 1872 tagagtctcc tcttgctcta ctcaatctac ccagagatgccccttagcac actcagaggg 1932 cagggaccat aggacccagg ttccaacccc attgtcagcaccccagccat gctgccatcc 1992 cttagcacac ctgctcgtcc cattcagctt accctcccagtcagccagaa tctgagggga 2052 gggcccccag agagccccct tccccatcag aagactgttgactgctttgc attttgggct 2112 cttctatata ttttgtaaaa taagaactat accagatctaataaaacaca atggctatgc 2172 aaaaaaaaaa aaaaaaaaa 2191 26 220 PRT Simiansp. 26 Met Arg Gly Gln Gly Arg Lys Glu Ser Leu Ser Asp Ser Arg Asp Leu 15 10 15 Asp Gly Ser Tyr Asp Gln Leu Thr Asp Ser Val Glu Asp Glu Phe Glu20 25 30 Leu Ser Thr Val Cys His Arg Pro Glu Gly Leu Glu Gln Leu Gln Glu35 40 45 Gln Thr Lys Phe Thr Arg Lys Glu Leu Gln Val Leu Tyr Arg Gly Phe50 55 60 Lys Asn Glu Cys Pro Ser Gly Ile Val Asn Glu Glu Asn Phe Lys Gln65 70 75 80 Ile Tyr Ser Gln Phe Phe Pro Gln Gly Asp Ser Ser Thr Tyr AlaThr 85 90 95 Phe Leu Phe Asn Ala Phe Asp Thr Asn His Asp Gly Ser Val SerPhe 100 105 110 Glu Asp Phe Val Ala Gly Leu Ser Val Ile Leu Arg Gly ThrVal Asp 115 120 125 Asp Arg Leu Asn Trp Ala Phe Asn Leu Tyr Asp Leu AsnLys Asp Gly 130 135 140 Cys Ile Thr Lys Glu Glu Met Leu Asp Ile Met LysSer Ile Tyr Asp 145 150 155 160 Met Met Gly Lys Tyr Thr Tyr Pro Ala LeuArg Glu Glu Ala Pro Arg 165 170 175 Glu His Val Glu Asn Phe Phe Gln LysMet Asp Arg Asn Lys Asp Gly 180 185 190 Val Val Thr Ile Glu Glu Phe IleGlu Ser Cys Gln Lys Asp Glu Asn 195 200 205 Ile Met Arg Ser Met Gln LeuPhe Asp Asn Val Ile 210 215 220 27 2057 DNA Simian sp. CDS (208)..(963)27 tgctgcccaa ggctcctgct cctgccccag gactctgagg tgggccctaa aacccagcgc 60tctctaaaga aaagccttgc cagcccctac tcccggcccc caaccccagc aggtcgctgc 120gccgccaggg ggcgctgtgt gagcgcccta ttctggccac ccggcgcccc ctcccacggc 180ccaggcggga gcggggcgcc gggggcc atg cgg ggc caa ggc aga aag gag agt 234Met Arg Gly Gln Gly Arg Lys Glu Ser 1 5 ttg tcc gaa tcc cga gat ctg gacggc tcc tat gac cag ctt acg ggc 282 Leu Ser Glu Ser Arg Asp Leu Asp GlySer Tyr Asp Gln Leu Thr Gly 10 15 20 25 cac cct cca ggg ccc agt aaa aaagcc ctg aag cag cgt ttc ctc aag 330 His Pro Pro Gly Pro Ser Lys Lys AlaLeu Lys Gln Arg Phe Leu Lys 30 35 40 ctg ctg ccg tgc tgc ggg ccc caa gccctg ccc tca gtc agt gaa aac 378 Leu Leu Pro Cys Cys Gly Pro Gln Ala LeuPro Ser Val Ser Glu Asn 45 50 55 agc gta gag gat gag ttt gaa tta tcc acggtg tgt cac cga cct gag 426 Ser Val Glu Asp Glu Phe Glu Leu Ser Thr ValCys His Arg Pro Glu 60 65 70 ggc ctg gaa caa ctc cag gaa cag acc aag ttcaca cgc aga gag ctg 474 Gly Leu Glu Gln Leu Gln Glu Gln Thr Lys Phe ThrArg Arg Glu Leu 75 80 85 cag gtc ctg tac cga ggc ttc aag aac gaa tgc cccagt ggg att gtc 522 Gln Val Leu Tyr Arg Gly Phe Lys Asn Glu Cys Pro SerGly Ile Val 90 95 100 105 aac gag gag aac ttc aag cag att tat tct cagttc ttt ccc caa gga 570 Asn Glu Glu Asn Phe Lys Gln Ile Tyr Ser Gln PhePhe Pro Gln Gly 110 115 120 gac tcc agc aac tat gct act ttt ctc ttc aatgcc ttt gac acc aac 618 Asp Ser Ser Asn Tyr Ala Thr Phe Leu Phe Asn AlaPhe Asp Thr Asn 125 130 135 cac gat ggc tct gtc agt ttt gag gac ttt gtggct ggt ttg tcg gtg 666 His Asp Gly Ser Val Ser Phe Glu Asp Phe Val AlaGly Leu Ser Val 140 145 150 att ctt cgg ggg acc ata gat gat aga ctg agctgg gct ttc aac tta 714 Ile Leu Arg Gly Thr Ile Asp Asp Arg Leu Ser TrpAla Phe Asn Leu 155 160 165 tat gac ctc aac aag gac ggc tgt atc aca aaggag gaa atg ctt gac 762 Tyr Asp Leu Asn Lys Asp Gly Cys Ile Thr Lys GluGlu Met Leu Asp 170 175 180 185 att atg aag tcc atc tat gac atg atg ggcaag tac aca tac cct gcc 810 Ile Met Lys Ser Ile Tyr Asp Met Met Gly LysTyr Thr Tyr Pro Ala 190 195 200 ctc cgg gag gag gcc cca aga gaa cac gtggag agc ttc ttc cag aag 858 Leu Arg Glu Glu Ala Pro Arg Glu His Val GluSer Phe Phe Gln Lys 205 210 215 atg gac agg aac aag gac ggc gtg gtg accatc gag gaa ttc atc gag 906 Met Asp Arg Asn Lys Asp Gly Val Val Thr IleGlu Glu Phe Ile Glu 220 225 230 tct tgt caa cag gac gag aac atc atg aggtcc atg cag ctc tca ccc 954 Ser Cys Gln Gln Asp Glu Asn Ile Met Arg SerMet Gln Leu Ser Pro 235 240 245 ctt ctc aac tgatacctag tgctgaggacacccctggtg tagggaccaa 1003 Leu Leu Asn 250 gtggttctcc accttctagtcccactctag aaaccacatt agacagaagg tctcctgcta 1063 tggtgctttc cccatccctaatctcttaga ttttcctcaa gactcccttc tcagagaaca 1123 cgctctgtcc atgtccccagctggcttctc agcctagcct ttgagggccc tgtggggagg 1183 cggggacaag aaagcagaaaagtcttggcc ccgagccagt ggttaggtcc taggaattgg 1243 ctggagtgga ggccagaaagcctgggcaga tgatgagagc ccagctgggc tgtcactgca 1303 ggttccgggg cctacagccctgggtcagca gagtatgagt tcccagactt tccagaaggt 1363 ccttagcaat gtcccagaaattcaccgtac acttctcagt gtcttaggag ggcccgggat 1423 ccagatgtct ggttcatccctgaatcctct ccctccttct tgctcgtatg gtgggagtgg 1483 tggccagggg aagatgagtggtgtcccgga tgatgcctgt caaggtccca cctcccctcc 1543 ggctgttctc atgacagctgtttggttctc catgacccct atctagatgt agaggcatgg 1603 agtgagtcag ggatttcccgaacttgagtt ttaccactcc tcctagtggc tgccttaggg 1663 gaatgggaag aacccagtgtgggggcaccc attagaatct ttgcccggct cctcacaatg 1723 ccctagggtc ccctagggtacccgctccct ctgtttagtc tacccagaga tgctcctgag 1783 ctcacctaga gggtagggacggtaggctcc aggtccaacc tctccaggtc agcaccctgc 1843 catgctgctg ctcctcattaacaaacctgc ttgtctcctc ctgcgcccct tctcagtcag 1903 ccagggtctg aggggaagggcctcccgttt ccccatccgt cagacatggt tgactgcttt 1963 gcattttggg ctcttctatctattttgtaa aataagacat cagatccaat aaaacacacg 2023 gctatgcaca aaaaaaaaaaaaaaaaaaaa aaaa 2057 28 252 PRT Simian sp. 28 Met Arg Gly Gln Gly ArgLys Glu Ser Leu Ser Glu Ser Arg Asp Leu 1 5 10 15 Asp Gly Ser Tyr AspGln Leu Thr Gly His Pro Pro Gly Pro Ser Lys 20 25 30 Lys Ala Leu Lys GlnArg Phe Leu Lys Leu Leu Pro Cys Cys Gly Pro 35 40 45 Gln Ala Leu Pro SerVal Ser Glu Asn Ser Val Glu Asp Glu Phe Glu 50 55 60 Leu Ser Thr Val CysHis Arg Pro Glu Gly Leu Glu Gln Leu Gln Glu 65 70 75 80 Gln Thr Lys PheThr Arg Arg Glu Leu Gln Val Leu Tyr Arg Gly Phe 85 90 95 Lys Asn Glu CysPro Ser Gly Ile Val Asn Glu Glu Asn Phe Lys Gln 100 105 110 Ile Tyr SerGln Phe Phe Pro Gln Gly Asp Ser Ser Asn Tyr Ala Thr 115 120 125 Phe LeuPhe Asn Ala Phe Asp Thr Asn His Asp Gly Ser Val Ser Phe 130 135 140 GluAsp Phe Val Ala Gly Leu Ser Val Ile Leu Arg Gly Thr Ile Asp 145 150 155160 Asp Arg Leu Ser Trp Ala Phe Asn Leu Tyr Asp Leu Asn Lys Asp Gly 165170 175 Cys Ile Thr Lys Glu Glu Met Leu Asp Ile Met Lys Ser Ile Tyr Asp180 185 190 Met Met Gly Lys Tyr Thr Tyr Pro Ala Leu Arg Glu Glu Ala ProArg 195 200 205 Glu His Val Glu Ser Phe Phe Gln Lys Met Asp Arg Asn LysAsp Gly 210 215 220 Val Val Thr Ile Glu Glu Phe Ile Glu Ser Cys Gln GlnAsp Glu Asn 225 230 235 240 Ile Met Arg Ser Met Gln Leu Ser Pro Leu LeuAsn 245 250 29 1904 DNA Rattus sp. CDS (1)..(675) 29 atg aac cac tgc cctcgc agg tgc cgg agc ccg ttg ggg cag gca gct 48 Met Asn His Cys Pro ArgArg Cys Arg Ser Pro Leu Gly Gln Ala Ala 1 5 10 15 cga tct ctc tac cagttg gta act ggg tcg ctg tcg cca gac agc gta 96 Arg Ser Leu Tyr Gln LeuVal Thr Gly Ser Leu Ser Pro Asp Ser Val 20 25 30 gag gat gag ttt gaa ttatcc acg gtg tgt cac cga cct gag ggc ctg 144 Glu Asp Glu Phe Glu Leu SerThr Val Cys His Arg Pro Glu Gly Leu 35 40 45 gaa caa ctc cag gaa cag accaag ttc aca cgc aga gag ctg cag gtc 192 Glu Gln Leu Gln Glu Gln Thr LysPhe Thr Arg Arg Glu Leu Gln Val 50 55 60 ctg tac cga ggc ttc aag aac gaatgc ccc agt ggg att gtc aac gag 240 Leu Tyr Arg Gly Phe Lys Asn Glu CysPro Ser Gly Ile Val Asn Glu 65 70 75 80 gag aac ttc aag cag att tat tctcag ttc ttt ccc caa gga gac tcc 288 Glu Asn Phe Lys Gln Ile Tyr Ser GlnPhe Phe Pro Gln Gly Asp Ser 85 90 95 agc aac tat gct act ttt ctc ttc aatgcc ttt gac acc aac cac gat 336 Ser Asn Tyr Ala Thr Phe Leu Phe Asn AlaPhe Asp Thr Asn His Asp 100 105 110 ggc tct gtc agt ttt gag gac ttt gtggct ggt ttg tcg gtg att ctt 384 Gly Ser Val Ser Phe Glu Asp Phe Val AlaGly Leu Ser Val Ile Leu 115 120 125 cgg ggg acc ata gat gat aga ctg agctgg gct ttc aac tta tat gac 432 Arg Gly Thr Ile Asp Asp Arg Leu Ser TrpAla Phe Asn Leu Tyr Asp 130 135 140 ctc aac aag gac ggc tgt atc aca aaggag gaa atg ctt gac att atg 480 Leu Asn Lys Asp Gly Cys Ile Thr Lys GluGlu Met Leu Asp Ile Met 145 150 155 160 aag tcc atc tat gac atg atg ggcaag tac aca tac cct gcc ctc cgg 528 Lys Ser Ile Tyr Asp Met Met Gly LysTyr Thr Tyr Pro Ala Leu Arg 165 170 175 gag gag gcc cca aga gaa cac gtggag agc ttc ttc cag aag atg gac 576 Glu Glu Ala Pro Arg Glu His Val GluSer Phe Phe Gln Lys Met Asp 180 185 190 agg aac aag gac ggc gtg gtg accatc gag gaa ttc atc gag tct tgt 624 Arg Asn Lys Asp Gly Val Val Thr IleGlu Glu Phe Ile Glu Ser Cys 195 200 205 caa cag gac gag aac atc atg aggtcc atg cag ctc ttt gat aat gtc 672 Gln Gln Asp Glu Asn Ile Met Arg SerMet Gln Leu Phe Asp Asn Val 210 215 220 atc tagctcccca gggagaggggttagtgtgtc ctagggtgac caggctgtag 725 Ile 225 tcctagtcca gacgaacctaaccctctctc tccaggcctg tcctcatctt acctgtaccc 785 tgggggctgt agggattcaatatcctgggg cttcagtagt ccagatccct gagctaagtc 845 acaaaagtag gcaagagtaggcaagctaaa tctgggggct tcccaacccc cgacagctct 905 caccccttct caactgatacctagtgctga ggacacccct ggtgtaggga ccaagtggtt 965 ctccaccttc tagtcccactctagaaacca cattagacag aaggtctcct gctatggtgc 1025 tttccccatc cctaatctcttagattttcc tcaagactcc cttctcagag aacacgctct 1085 gtccatgtcc ccagctggcttctcagccta gcctttgagg gccctgtggg gaggcgggga 1145 caagaaagca gaaaagtcttggccccgagc tagtggttag gtcctaggaa ttggctggag 1205 tggaggccag aaagcctgggcagatgatga gagcccagct gggctgtcac tgcaggttcc 1265 agggcctaca gccctgggtcagcagagtat gagttcccag actttccaga aggtccttag 1325 caatgtccca gaaattcaccatacacttct cagtgtcccg gatgatgcct gtcaaggtcc 1385 cacctcccct ccggctgttctcatgacagc tgtttggttc tccatgaccc ctatctagat 1445 gtagaggcat ggagtgagtcagggatttcc cgaacttgag ttttaccact cctcctagtg 1505 gctgccttag gggaatgggaagaacccagt gtgggggcac ccattagaat ctttgcccgg 1565 ttcctcacaa tgccctagggtcccctaggg tacccgctcc ctctgtttag tctacccaga 1625 gatgctcctg agctcacctagagggtaggg acggtaggct ccaggtccaa cctctccagg 1685 tcagcaccct gccatgctgctgctcctcat taacaaacct gcttgtctcc tcctgcgccc 1745 cttctcagtc agccagggtctgaggggaag ggcctcccgt ttccccatcc gtcagacatg 1805 gttgactgct ttgcattttgggctcttcta tctattttgt aaaataagac atcagatcca 1865 ataaaacaca cggctatgcacaaaaaaaaa aaaaaaaaa 1904 30 225 PRT Rattus sp. 30 Met Asn His Cys ProArg Arg Cys Arg Ser Pro Leu Gly Gln Ala Ala 1 5 10 15 Arg Ser Leu TyrGln Leu Val Thr Gly Ser Leu Ser Pro Asp Ser Val 20 25 30 Glu Asp Glu PheGlu Leu Ser Thr Val Cys His Arg Pro Glu Gly Leu 35 40 45 Glu Gln Leu GlnGlu Gln Thr Lys Phe Thr Arg Arg Glu Leu Gln Val 50 55 60 Leu Tyr Arg GlyPhe Lys Asn Glu Cys Pro Ser Gly Ile Val Asn Glu 65 70 75 80 Glu Asn PheLys Gln Ile Tyr Ser Gln Phe Phe Pro Gln Gly Asp Ser 85 90 95 Ser Asn TyrAla Thr Phe Leu Phe Asn Ala Phe Asp Thr Asn His Asp 100 105 110 Gly SerVal Ser Phe Glu Asp Phe Val Ala Gly Leu Ser Val Ile Leu 115 120 125 ArgGly Thr Ile Asp Asp Arg Leu Ser Trp Ala Phe Asn Leu Tyr Asp 130 135 140Leu Asn Lys Asp Gly Cys Ile Thr Lys Glu Glu Met Leu Asp Ile Met 145 150155 160 Lys Ser Ile Tyr Asp Met Met Gly Lys Tyr Thr Tyr Pro Ala Leu Arg165 170 175 Glu Glu Ala Pro Arg Glu His Val Glu Ser Phe Phe Gln Lys MetAsp 180 185 190 Arg Asn Lys Asp Gly Val Val Thr Ile Glu Glu Phe Ile GluSer Cys 195 200 205 Gln Gln Asp Glu Asn Ile Met Arg Ser Met Gln Leu PheAsp Asn Val 210 215 220 Ile 225 31 2841 DNA Homo sapiens CDS (1)..(768)31 atg cag ccg gct aag gaa gtg aca aag gcg tcg gac ggc agc ctc ctg 48Met Gln Pro Ala Lys Glu Val Thr Lys Ala Ser Asp Gly Ser Leu Leu 1 5 1015 ggg gac ctc ggg cac aca cca ctt agc aag aag gag ggt atc aag tgg 96Gly Asp Leu Gly His Thr Pro Leu Ser Lys Lys Glu Gly Ile Lys Trp 20 25 30cag agg ccg agg ctc agc cgc cag gct ttg atg aga tgc tgc ctg gtc 144 GlnArg Pro Arg Leu Ser Arg Gln Ala Leu Met Arg Cys Cys Leu Val 35 40 45 aagtgg atc ctg tcc agc aca gcc cca cag ggc tca gat agc agc gac 192 Lys TrpIle Leu Ser Ser Thr Ala Pro Gln Gly Ser Asp Ser Ser Asp 50 55 60 agt gagctg gag ctg tcc acg gtg cgc cac cag cca gag ggg ctg gac 240 Ser Glu LeuGlu Leu Ser Thr Val Arg His Gln Pro Glu Gly Leu Asp 65 70 75 80 cag ctgcag gcc cag acc aag ttc acc aag aag gag ctg cag tct ctc 288 Gln Leu GlnAla Gln Thr Lys Phe Thr Lys Lys Glu Leu Gln Ser Leu 85 90 95 tac agg ggcttt aag aat gag tgt ccc acg ggc ctg gtg gac gaa gac 336 Tyr Arg Gly PheLys Asn Glu Cys Pro Thr Gly Leu Val Asp Glu Asp 100 105 110 acc ttc aaactc att tac gcg cag ttc ttc cct cag gga gat gcc acc 384 Thr Phe Lys LeuIle Tyr Ala Gln Phe Phe Pro Gln Gly Asp Ala Thr 115 120 125 acc tat gcacac ttc ctc ttc aac gcc ttt gat gcg gac ggg aac ggg 432 Thr Tyr Ala HisPhe Leu Phe Asn Ala Phe Asp Ala Asp Gly Asn Gly 130 135 140 gcc atc cacttt gag gac ttt gtg gtt ggc ctc tcc atc ctg ctg cgg 480 Ala Ile His PheGlu Asp Phe Val Val Gly Leu Ser Ile Leu Leu Arg 145 150 155 160 ggc acagtc cac gag aag ctc aag tgg gcc ttt aat ctc tac gac att 528 Gly Thr ValHis Glu Lys Leu Lys Trp Ala Phe Asn Leu Tyr Asp Ile 165 170 175 aac aaggat ggc tac atc acc aaa gag gag atg ctg gcc atc atg aag 576 Asn Lys AspGly Tyr Ile Thr Lys Glu Glu Met Leu Ala Ile Met Lys 180 185 190 tcc atctat gac atg atg ggc cgc cac acc tac ccc atc ctg cgg gag 624 Ser Ile TyrAsp Met Met Gly Arg His Thr Tyr Pro Ile Leu Arg Glu 195 200 205 gac gcgccg gcg gag cac gtg gag agg ttc ttc gag aaa atg gac cgg 672 Asp Ala ProAla Glu His Val Glu Arg Phe Phe Glu Lys Met Asp Arg 210 215 220 aac caggat ggg gta gtg acc att gaa gag ttc ctg gag gcc tgt cag 720 Asn Gln AspGly Val Val Thr Ile Glu Glu Phe Leu Glu Ala Cys Gln 225 230 235 240 aaggat gag aac atc atg agc tcc atg cag ctg ttt gag aat gtc atc 768 Lys AspGlu Asn Ile Met Ser Ser Met Gln Leu Phe Glu Asn Val Ile 245 250 255taggacacgt ccaaaggagt gcatggccac agccacctcc acccccaaga aacctccatc 828ctgccaggag cagcctccaa gaaactttta aaaaatagat ttgcaaaaag tgaacagatt 888gctacacaca cacacacaca cacacacaca cacacacaca cacagccatt catctgggct 948ggcagagggg acagagttca gggaggggct gagtctggct aggggccgag tccaggagcc 1008ccagccagcc cttcccaggc cagcgaggcg aggctgcctc tgggtgagtg gctgacagag 1068caggtctgca ggccaccagc tgctggatgt caccaagaag gggctcgagt gcccctgcag 1128gggagggtcc aatctccggt gtgagcccac ctcgtcccgt tctccattct gctttcttgc 1188cacacagtgg gccggcccca ggctcccctg gtctcctccc cgtagccact ctctgcccac 1248tacctatgct tctagaaagc ccctcacctc aggaccccag agggaccagc tggggggcag 1308gggggagagg gggtaatgga ggccaagcct gcagctttct ggaaattctt ccctgggggt 1368cccaggatcc cctgctactc cactgacctg gaagagctgg gtaccaggcc acccactgtg 1428gggcaagcct gagtggtgag gggccactgg gccccattct ccctccatgg caggaaggcg 1488ggggatttca agtttaggga ttgggtcgtg gtggagaatc tgagggcact ctctgccagc 1548tccacagggt gggatgagcc tctccttgcc ccagtcctgg ttcagtggga atgcagtggg 1608tggggctgta cacaccctcc agcacagact gttccctcca aggtcctctt aggtcccggg 1668aggaacgtgg ttcagagact ggcagccagg gagcccgggg cagagctcag aggagtctgg 1728gaaggggcgt gtccctcctc ttcctgtagt gcccctccca tggcccagca gcttggctga 1788gccccctctc ctgaagcagt gtcgccgtcc ctctgccttg cacaaaaagc acaagcattc 1848cttagcagct caggcgcagc cctagtggga gcccagcaca ctgcttctcg gaggccaggc 1908cctcctgctg gctgaggctt gggcccagta gccccaatat ggtggccctg gggaagaggc 1968cttgggggtc tgctctgtgc ctgggatcag tggggcccca aagcccagcc cggctgacca 2028acattcaaaa gcacaaaccc tggggactct gcttggctgt cccctccatc tggggatgga 2088gaatgccagc ccaaagctgg agccaatggt gagggctgag agggctgtgg ctgggtggtc 2148agcagaaacc cccaggagga gagagatgct gctcccgcct gattggggcc tcacccagaa 2208ggaacccggt cccaggccgc atggcccctc caggaacatt cccacataat acattccatc 2268acagccagcc cagctccact cagggctggc ccggggagtc cccgtgtgcc ccaagaggct 2328agccccaggg tgagcagggc cctcagagga aaggcagtat ggcggaggcc atgggggccc 2388ctcggcattc acacacagcc tggcctcccc tgcggagctg catggacgcc tggctccagg 2448ctccaggctg actgggggcc tctgcctcca ggagggcatc agctttccct ggctcaggga 2508tcttctccct cccctcaccc gctgcccagc cctcccagct ggtgtcactc tgcctctaag 2568gccaaggcct caggagagca tcaccaccac acccctgccg gccttggcct tggggccaga 2628ctggctgcac agcccaacca ggaggggtct gcctcccacg ctgggacaca gaccggccgc 2688atgtctgcat ggcagaagcg tctcccttgg ccacggcctg ggagggtggt tcctgttctc 2748agcatccact aatattcagt cctgtatatt ttaataaaat aaacttgaca aaggaaaaaa 2808aaaaaaaaaa aattcctgcg gccgcgttct cca 2841 32 256 PRT Homo sapiens 32 MetGln Pro Ala Lys Glu Val Thr Lys Ala Ser Asp Gly Ser Leu Leu 1 5 10 15Gly Asp Leu Gly His Thr Pro Leu Ser Lys Lys Glu Gly Ile Lys Trp 20 25 30Gln Arg Pro Arg Leu Ser Arg Gln Ala Leu Met Arg Cys Cys Leu Val 35 40 45Lys Trp Ile Leu Ser Ser Thr Ala Pro Gln Gly Ser Asp Ser Ser Asp 50 55 60Ser Glu Leu Glu Leu Ser Thr Val Arg His Gln Pro Glu Gly Leu Asp 65 70 7580 Gln Leu Gln Ala Gln Thr Lys Phe Thr Lys Lys Glu Leu Gln Ser Leu 85 9095 Tyr Arg Gly Phe Lys Asn Glu Cys Pro Thr Gly Leu Val Asp Glu Asp 100105 110 Thr Phe Lys Leu Ile Tyr Ala Gln Phe Phe Pro Gln Gly Asp Ala Thr115 120 125 Thr Tyr Ala His Phe Leu Phe Asn Ala Phe Asp Ala Asp Gly AsnGly 130 135 140 Ala Ile His Phe Glu Asp Phe Val Val Gly Leu Ser Ile LeuLeu Arg 145 150 155 160 Gly Thr Val His Glu Lys Leu Lys Trp Ala Phe AsnLeu Tyr Asp Ile 165 170 175 Asn Lys Asp Gly Tyr Ile Thr Lys Glu Glu MetLeu Ala Ile Met Lys 180 185 190 Ser Ile Tyr Asp Met Met Gly Arg His ThrTyr Pro Ile Leu Arg Glu 195 200 205 Asp Ala Pro Ala Glu His Val Glu ArgPhe Phe Glu Lys Met Asp Arg 210 215 220 Asn Gln Asp Gly Val Val Thr IleGlu Glu Phe Leu Glu Ala Cys Gln 225 230 235 240 Lys Asp Glu Asn Ile MetSer Ser Met Gln Leu Phe Glu Asn Val Ile 245 250 255 33 442 DNA Rattussp. CDS (1)..(327) 33 ttt gag gac ttt gtg gtt ggg ctc tcc atc ctg cttcga ggg acc gtc 48 Phe Glu Asp Phe Val Val Gly Leu Ser Ile Leu Leu ArgGly Thr Val 1 5 10 15 cat gag aag ctc aag tgg gcc ttc aat ctc tac gacatc aac aag gac 96 His Glu Lys Leu Lys Trp Ala Phe Asn Leu Tyr Asp IleAsn Lys Asp 20 25 30 ggt tac atc acc aaa gag gag atg ctg gcc atc atg aagtcc atc tac 144 Gly Tyr Ile Thr Lys Glu Glu Met Leu Ala Ile Met Lys SerIle Tyr 35 40 45 gac atg atg ggc cgc cac acc tac cct atc ctg cgg gag gacgca cct 192 Asp Met Met Gly Arg His Thr Tyr Pro Ile Leu Arg Glu Asp AlaPro 50 55 60 ctg gag cat gtg gag agg ttc ttc cag aaa atg gac agg aac caggat 240 Leu Glu His Val Glu Arg Phe Phe Gln Lys Met Asp Arg Asn Gln Asp65 70 75 80 gga gta gtg act att gat gaa ttt ctg gag act tgt cag aag gacgag 288 Gly Val Val Thr Ile Asp Glu Phe Leu Glu Thr Cys Gln Lys Asp Glu85 90 95 aac atc atg agc tcc atg cag ctg ttt gag aac gtc atc taggacatgt337 Asn Ile Met Ser Ser Met Gln Leu Phe Glu Asn Val Ile 100 105aggaggggac cctgggtggc catgggttct caacccagag aagcctcaat cctgacagga 397gaagcctcta tgagaaacat ttttctaata tatttgcaaa aagtg 442 34 109 PRT Rattussp. 34 Phe Glu Asp Phe Val Val Gly Leu Ser Ile Leu Leu Arg Gly Thr Val 15 10 15 His Glu Lys Leu Lys Trp Ala Phe Asn Leu Tyr Asp Ile Asn Lys Asp20 25 30 Gly Tyr Ile Thr Lys Glu Glu Met Leu Ala Ile Met Lys Ser Ile Tyr35 40 45 Asp Met Met Gly Arg His Thr Tyr Pro Ile Leu Arg Glu Asp Ala Pro50 55 60 Leu Glu His Val Glu Arg Phe Phe Gln Lys Met Asp Arg Asn Gln Asp65 70 75 80 Gly Val Val Thr Ile Asp Glu Phe Leu Glu Thr Cys Gln Lys AspGlu 85 90 95 Asn Ile Met Ser Ser Met Gln Leu Phe Glu Asn Val Ile 100 10535 2644 DNA Mus musculus CDS (49)..(816) 35 cgggctgcaa agcgggaagattagtgacgg tccctttcag cagcagag atg cag agg 57 Met Gln Arg 1 acc aag gaagcc gtg aag gca tca gat ggc aac ctc ctg gga gat cct 105 Thr Lys Glu AlaVal Lys Ala Ser Asp Gly Asn Leu Leu Gly Asp Pro 5 10 15 ggg cgc ata ccactg agc aag agg gaa agc atc aag tgg caa agg cca 153 Gly Arg Ile Pro LeuSer Lys Arg Glu Ser Ile Lys Trp Gln Arg Pro 20 25 30 35 cgg ttc acc cgccag gcc ctg atg cgt tgc tgc tta atc aag tgg atc 201 Arg Phe Thr Arg GlnAla Leu Met Arg Cys Cys Leu Ile Lys Trp Ile 40 45 50 ctg tcc agt gct gcccca caa ggc tca gac agc agt gac agt gaa ctg 249 Leu Ser Ser Ala Ala ProGln Gly Ser Asp Ser Ser Asp Ser Glu Leu 55 60 65 gag tta tcc acg gtg cgccat cag cca gag ggc ttg gac cag cta caa 297 Glu Leu Ser Thr Val Arg HisGln Pro Glu Gly Leu Asp Gln Leu Gln 70 75 80 gct cag acc aag ttc acc aagaag gag ctg cag tcc ctt tac cga ggc 345 Ala Gln Thr Lys Phe Thr Lys LysGlu Leu Gln Ser Leu Tyr Arg Gly 85 90 95 ttc aag aat gag tgt ccc aca ggcctg gtg gat gaa gac acc ttc aaa 393 Phe Lys Asn Glu Cys Pro Thr Gly LeuVal Asp Glu Asp Thr Phe Lys 100 105 110 115 ctc att tat tcc cag ttc ttccct cag gga gat gcc acc acc tat gca 441 Leu Ile Tyr Ser Gln Phe Phe ProGln Gly Asp Ala Thr Thr Tyr Ala 120 125 130 cac ttc ctc ttc aat gcc tttgat gct gat ggg aac ggg gcc atc cac 489 His Phe Leu Phe Asn Ala Phe AspAla Asp Gly Asn Gly Ala Ile His 135 140 145 ttt gag gac ttt gtg gtt gggctc tcc atc ctg ctt cga ggg acg gtc 537 Phe Glu Asp Phe Val Val Gly LeuSer Ile Leu Leu Arg Gly Thr Val 150 155 160 cat gag aag ctc aag tgg gccttc aat ctc tat gac att aac aag gat 585 His Glu Lys Leu Lys Trp Ala PheAsn Leu Tyr Asp Ile Asn Lys Asp 165 170 175 ggt tgc atc acc aag gag gagatg ctg gcc atc atg aag tcc atc tac 633 Gly Cys Ile Thr Lys Glu Glu MetLeu Ala Ile Met Lys Ser Ile Tyr 180 185 190 195 gac atg atg ggc cgc cacacc tac ccc atc ctg cgg gag gat gca ccc 681 Asp Met Met Gly Arg His ThrTyr Pro Ile Leu Arg Glu Asp Ala Pro 200 205 210 ctg gag cat gtg gag aggttc ttt cag aaa atg gac agg aac cag gat 729 Leu Glu His Val Glu Arg PhePhe Gln Lys Met Asp Arg Asn Gln Asp 215 220 225 gga gtg gtg acc att gatgaa ttt ctg gag act tgt cag aag gat gag 777 Gly Val Val Thr Ile Asp GluPhe Leu Glu Thr Cys Gln Lys Asp Glu 230 235 240 aac atc atg aac tcc atgcag ctg ttt gag aac gtc atc taggacatgt 826 Asn Ile Met Asn Ser Met GlnLeu Phe Glu Asn Val Ile 245 250 255 gggaggggac cccagtggtc attgcttctcaacccagaga agcctcaatc ctgacaggag 886 aagcctctat gagaaacatt tttctaatatatttgcaaaa agtgagcagt ttacttccaa 946 gacacagcca ccgtcacaca cagacacagacatacagaca cacacacaca cacacacaca 1006 tggttcctct ggcttggcca aggaagtggcagccagaagg cacccccgcc tattcctagg 1066 tcaataaaaa aggctgcctc tgggatggccagccctggct agatgttacc cacaaggaac 1126 tcagagatcg agaggaccag gtctacaaagctaaggtccc tgtgtctttt ctaccactcg 1186 ggagatcaaa ctactccctg cctatggacccatgctctta ggaagctccc agaaactcca 1246 aggggacaaa gaggggagag gtctataggaagaaatggtt ttggaagctg ggcttgcagc 1306 cttatgctaa tgatcacctg gggtcctggaacccgagtgc caggctacct actatgccgt 1366 gagcttagat agtgaggggc cattggactaagacctcctg taagagtggg gcaggattga 1426 ggtttttgga gaaactgagg aaacaatttgtccataccac tgggtgaaga ctgctggcca 1486 gtgggaatgt ggctggtgga gatttcccaacttccagcac caggatggcc tctccaaggt 1546 cctctttgat tccctgggga gatcacctggctcatagact gacaaccagg gaactgggct 1606 gaaatgggag gtctggtagg gggcatccccctccttttcc ctggccactt gccacccagt 1666 tccttaacac agtggatcgg ccacacctctgtggctgccc ttgaacagac tcatcccgac 1726 caagacaaaa aagcacaaac tcctagcagctcaggccaag cccacaaggg aaggcctggg 1786 tccctgcagc cctgattcag tggccgaggaagacgctcag acatccatcc tgtacctcgg 1846 agccttgggg gtctcacagc cctttcccagcccagctcgc caacattcta aagcacaaac 1906 ctgcggattc tgcttgcttg ggctgcgccctggggattga aggccactgt taaccctaag 1966 ctggagctag ccctgagggc tggggacctgtgaccaggca acaggtcagc agaccctcag 2026 gaggagagag agctgttcct gcctccccaggcctcgccca gaaggaacag tgtcccaaga 2086 agcatgtttc ctggaggaac atccccacaaaagtacattc catcatctga agcccggtct 2146 ctgctcaggc ctgcctctga aagtccacgtgtgttcccca gaaggccagc cccaagataa 2206 gggaggtcct tagaggaagg acagggtgacaacaccccta tacacaggtg gaccccccct 2266 ctgaggactg tactgacccc atctccatcctgaccggggc cttcctttac ccgatctaca 2326 gaccaccagt tctccctggc tcagggaccccctgtccccc agtctgactc ttcccatcga 2386 ggtccctgtc ttgtgaaaag ccaaggccacgggaaaaggc caccactcta acctgctgca 2446 tcccttagcc tctggctgca cgcccaacctggaggggtct gtcccctttg cagggacaca 2506 gactggccgc atgtccgcat ggcagaagcgtctcccttgg gtgcagcctg gaagggtggt 2566 ttctgtctca gcgcccacca atattcagtcctatatattt taataaaaga aacttgacaa 2626 aggaaaaaaa aaaaaaaa 2644 36 256PRT Mus musculus 36 Met Gln Arg Thr Lys Glu Ala Val Lys Ala Ser Asp GlyAsn Leu Leu 1 5 10 15 Gly Asp Pro Gly Arg Ile Pro Leu Ser Lys Arg GluSer Ile Lys Trp 20 25 30 Gln Arg Pro Arg Phe Thr Arg Gln Ala Leu Met ArgCys Cys Leu Ile 35 40 45 Lys Trp Ile Leu Ser Ser Ala Ala Pro Gln Gly SerAsp Ser Ser Asp 50 55 60 Ser Glu Leu Glu Leu Ser Thr Val Arg His Gln ProGlu Gly Leu Asp 65 70 75 80 Gln Leu Gln Ala Gln Thr Lys Phe Thr Lys LysGlu Leu Gln Ser Leu 85 90 95 Tyr Arg Gly Phe Lys Asn Glu Cys Pro Thr GlyLeu Val Asp Glu Asp 100 105 110 Thr Phe Lys Leu Ile Tyr Ser Gln Phe PhePro Gln Gly Asp Ala Thr 115 120 125 Thr Tyr Ala His Phe Leu Phe Asn AlaPhe Asp Ala Asp Gly Asn Gly 130 135 140 Ala Ile His Phe Glu Asp Phe ValVal Gly Leu Ser Ile Leu Leu Arg 145 150 155 160 Gly Thr Val His Glu LysLeu Lys Trp Ala Phe Asn Leu Tyr Asp Ile 165 170 175 Asn Lys Asp Gly CysIle Thr Lys Glu Glu Met Leu Ala Ile Met Lys 180 185 190 Ser Ile Tyr AspMet Met Gly Arg His Thr Tyr Pro Ile Leu Arg Glu 195 200 205 Asp Ala ProLeu Glu His Val Glu Arg Phe Phe Gln Lys Met Asp Arg 210 215 220 Asn GlnAsp Gly Val Val Thr Ile Asp Glu Phe Leu Glu Thr Cys Gln 225 230 235 240Lys Asp Glu Asn Ile Met Asn Ser Met Gln Leu Phe Glu Asn Val Ile 245 250255 37 531 DNA Homo sapiens CDS (1)..(336) At position 495, n=any aminoacid 37 cac gag gtg gaa agc att tcg gct cag ctg gag gag gcc agc tct aca48 His Glu Val Glu Ser Ile Ser Ala Gln Leu Glu Glu Ala Ser Ser Thr 1 510 15 ggc ggt ttc ctg tac gct cag aac agc acc aag cgc agc att aaa gag 96Gly Gly Phe Leu Tyr Ala Gln Asn Ser Thr Lys Arg Ser Ile Lys Glu 20 25 30cgg ctc atg aag ctc ttg ccc tgc tca gct gcc aaa acg tcg tct cct 144 ArgLeu Met Lys Leu Leu Pro Cys Ser Ala Ala Lys Thr Ser Ser Pro 35 40 45 gctatt caa aac agc gtg gaa gat gaa ctg gag atg gcc acc gtc agg 192 Ala IleGln Asn Ser Val Glu Asp Glu Leu Glu Met Ala Thr Val Arg 50 55 60 cat cggccc gaa gcc ctt gag ctt ctg gaa gcc cag agc aaa ttt acc 240 His Arg ProGlu Ala Leu Glu Leu Leu Glu Ala Gln Ser Lys Phe Thr 65 70 75 80 aag aaagag ctt cag atc ctt tac aga gga ttt aag aac gta aga act 288 Lys Lys GluLeu Gln Ile Leu Tyr Arg Gly Phe Lys Asn Val Arg Thr 85 90 95 ttc ttt ttgact tta cct tca cac aat tcc cag agg agc att gag aaa 336 Phe Phe Leu ThrLeu Pro Ser His Asn Ser Gln Arg Ser Ile Glu Lys 100 105 110 tgagaggaaaagggggaaaa tatcccattc tatgagaagc cccatcatat gtatatttca 396 tactgatccttcccagatag gaatataatc agtatctgtg gactttgaat ctctgtggca 456 cacccatgctggcatactgt aattgcccat taaacaaana gtttttgaga aaaaaaaaaa 516 aaaaaaaaaaaaaaa 531 38 112 PRT Homo sapiens 38 His Glu Val Glu Ser Ile Ser Ala GlnLeu Glu Glu Ala Ser Ser Thr 1 5 10 15 Gly Gly Phe Leu Tyr Ala Gln AsnSer Thr Lys Arg Ser Ile Lys Glu 20 25 30 Arg Leu Met Lys Leu Leu Pro CysSer Ala Ala Lys Thr Ser Ser Pro 35 40 45 Ala Ile Gln Asn Ser Val Glu AspGlu Leu Glu Met Ala Thr Val Arg 50 55 60 His Arg Pro Glu Ala Leu Glu LeuLeu Glu Ala Gln Ser Lys Phe Thr 65 70 75 80 Lys Lys Glu Leu Gln Ile LeuTyr Arg Gly Phe Lys Asn Val Arg Thr 85 90 95 Phe Phe Leu Thr Leu Pro SerHis Asn Ser Gln Arg Ser Ile Glu Lys 100 105 110 39 2176 DNA Homo sapiensCDS (2)..(124) 39 t gaa agg ttc ttc gag aaa atg gac cgg aac cag gat ggggta gtg acc 49 Glu Arg Phe Phe Glu Lys Met Asp Arg Asn Gln Asp Gly ValVal Thr 1 5 10 15 att gaa gag ttc ctg gag gcc tgt cag aag gat gag aacatc atg agc 97 Ile Glu Glu Phe Leu Glu Ala Cys Gln Lys Asp Glu Asn IleMet Ser 20 25 30 tcc atg cag ctg ttt gag aat gtc atc taggacacgtccaaaggagt 144 Ser Met Gln Leu Phe Glu Asn Val Ile 35 40 gcatggccacagccacctcc acccccaaga aacctccatc ctgccaggag cagcctccaa 204 gaaacttttaaaaaatagat ttgcaaaaag tgaacagatt gctacacaca cacacacaca 264 cacacacacacacacacaca cacagccatt catctgggct ggcagagggg acagagttca 324 gggaggggctgagtctggct aggggccgag tccaggagcc ccagccagcc cttcccaggc 384 cagcgaggcgaggctgcctc tgggtgagtg gctgacagag caggtctgca ggccaccagc 444 tgctggatgtcaccaagaag gggctcgagt gcccctgcag gggagggtcc aatctccggt 504 gtgagcccacctcgtcccgt tctccattct gctttcttgc cacacagtgg gccggcccca 564 ggctcccctggtctcctccc cgtagccact ctctgcccac tacctatgct tctagaaagc 624 ccctcacctcaggaccccag agggaccagc tggggggcag gggggagagg gggtaatgga 684 ggccaagcctgcagctttct ggaaattctt ccctgggggt cccaggatcc cctgctactc 744 cactgacctggaagagctgg gtaccaggcc acccactgtg gggcaagcct gagtggtgag 804 gggccactgggccccattct ccctccatgg caggaaggcg ggggatttca agtttaggga 864 ttgggtcgtggtggagaatc tgagggcact ctctgccagc tccacagggt gggatgagcc 924 tctccttgccccagtcctgg ttcagtggga atgcagtggg tggggctgta cacaccctcc 984 agcacagactgttccctcca aggtcctctt aggtcccggg aggaacgtgg ttcagagact 1044 ggcagccagggagcccgggg cagagctcag aggagtctgg gaaggggcgt gtccctcctc 1104 ttcctgtagtgcccctccca tggcccagca gcttggctga gccccctctc ctgaagcagt 1164 gtcgccgtccctctgccttg cacaaaaagc acaagcattc cttagcagct caggcgcagc 1224 cctagtgggagcccagcaca ctgcttctcg gaggccaggc cctcctgctg gctgaggctt 1284 gggcccagtagccccaatat ggtggccctg gggaagaggc cttgggggtc tgctctgtgc 1344 ctgggatcagtggggcccca aagcccagcc cggctgacca acattcaaaa gcacaaaccc 1404 tggggactctgcttggctgt cccctccatc tggggatgga gaatgccagc ccaaagctgg 1464 agccaatggtgagggctgag agggctgtgg ctgggtggtc agcagaaacc cccaggagga 1524 gagagatgctgctcccgcct gattggggcc tcacccagaa ggaacccggt cccaggccgc 1584 atggcccctccaggaacatt cccacataat acattccatc acagccagcc cagctccact 1644 cagggctggcccggggagtc cccgtgtgcc ccaagaggct agccccaggg tgagcagggc 1704 cctcagaggaaaggcagtat ggcggaggcc atgggggccc ctcggcattc acacacagcc 1764 tggcctcccctgcggagctg catggacgcc tggctccagg ctccaggctg actgggggcc 1824 tctgcctccaggagggcatc agctttccct ggctcaggga tcttctccct cccctcaccc 1884 gctgcccagccctcccagct ggtgtcactc tgcctctaag gccaaggcct caggagagca 1944 tcaccaccacacccctgccg gccttggcct tggggccaga ctggctgcac agcccaacca 2004 ggaggggtctgcctcccacg ctgggacaca gaccggccgc atgtctgcat ggcagaagcg 2064 tctcccttggccacggcctg ggagggtggt tcctgttctc agcatccact aatattcagt 2124 cctgtatattttaataaaat aaacttgaca aaggaaaaaa aaaaaaaaaa aa 2176 40 41 PRT Homosapiens 40 Glu Arg Phe Phe Glu Lys Met Asp Arg Asn Gln Asp Gly Val ValThr 1 5 10 15 Ile Glu Glu Phe Leu Glu Ala Cys Gln Lys Asp Glu Asn IleMet Ser 20 25 30 Ser Met Gln Leu Phe Glu Asn Val Ile 35 40 41 2057 DNARattus sp. CDS (208)..(963) 41 tgctgcccaa ggctcctgct cctgccccaggactctgagg tgggccctaa aacccagcgc 60 tctctaaaga aaagccttgc cagcccctactcccggcccc caaccccagc aggtcgctgc 120 gccgccaggg ggcgctgtgt gagcgccctattctggccac ccggcgcccc ctcccacggc 180 ccaggcggga gcggggcgcc gggggcc atgcgg ggc caa ggc aga aag gag agt 234 Met Arg Gly Gln Gly Arg Lys Glu Ser1 5 ttg tcc gaa tcc cga gat ctg gac ggc tcc tat gac cag ctt acg ggc 282Leu Ser Glu Ser Arg Asp Leu Asp Gly Ser Tyr Asp Gln Leu Thr Gly 10 15 2025 cac cct cca ggg ccc agt aaa aaa gcc ctg aag cag cgt ttc ctc aag 330His Pro Pro Gly Pro Ser Lys Lys Ala Leu Lys Gln Arg Phe Leu Lys 30 35 40ctg ctg ccg tgc tgc ggg ccc caa gcc ctg ccc tca gtc agt gaa aac 378 LeuLeu Pro Cys Cys Gly Pro Gln Ala Leu Pro Ser Val Ser Glu Asn 45 50 55 agcgta gag gat gag ttt gaa tta tcc acg gtg tgt cac cga cct gag 426 Ser ValGlu Asp Glu Phe Glu Leu Ser Thr Val Cys His Arg Pro Glu 60 65 70 ggc ctggaa caa ctc cag gaa cag acc aag ttc aca cgc aga gag ctg 474 Gly Leu GluGln Leu Gln Glu Gln Thr Lys Phe Thr Arg Arg Glu Leu 75 80 85 cag gtc ctgtac cga ggc ttc aag aac gaa tgc ccc agt ggg att gtc 522 Gln Val Leu TyrArg Gly Phe Lys Asn Glu Cys Pro Ser Gly Ile Val 90 95 100 105 aac gaggag aac ttc aag cag att tat tct cag ttc ttt ccc caa gga 570 Asn Glu GluAsn Phe Lys Gln Ile Tyr Ser Gln Phe Phe Pro Gln Gly 110 115 120 gac tccagc aac tat gct act ttt ctc ttc aat gcc ttt gac acc aac 618 Asp Ser SerAsn Tyr Ala Thr Phe Leu Phe Asn Ala Phe Asp Thr Asn 125 130 135 cac gatggc tct gtc agt ttt gag gac ttt gtg gct ggt ttg tcg gtg 666 His Asp GlySer Val Ser Phe Glu Asp Phe Val Ala Gly Leu Ser Val 140 145 150 att cttcgg ggg acc ata gat gat aga ctg agc tgg gct ttc aac tta 714 Ile Leu ArgGly Thr Ile Asp Asp Arg Leu Ser Trp Ala Phe Asn Leu 155 160 165 tat gacctc aac aag gac ggc tgt atc aca aag gag gaa atg ctt gac 762 Tyr Asp LeuAsn Lys Asp Gly Cys Ile Thr Lys Glu Glu Met Leu Asp 170 175 180 185 attatg aag tcc atc tat gac atg atg ggc aag tac aca tac cct gcc 810 Ile MetLys Ser Ile Tyr Asp Met Met Gly Lys Tyr Thr Tyr Pro Ala 190 195 200 ctccgg gag gag gcc cca aga gaa cac gtg gag agc ttc ttc cag aag 858 Leu ArgGlu Glu Ala Pro Arg Glu His Val Glu Ser Phe Phe Gln Lys 205 210 215 atggac agg aac aag gac ggc gtg gtg acc atc gag gaa ttc atc gag 906 Met AspArg Asn Lys Asp Gly Val Val Thr Ile Glu Glu Phe Ile Glu 220 225 230 tcttgt caa cag gac gag aac atc atg agg tcc atg cag ctc tca ccc 954 Ser CysGln Gln Asp Glu Asn Ile Met Arg Ser Met Gln Leu Ser Pro 235 240 245 cttctc aac tgatacctag tgctgaggac acccctggtg tagggaccaa 1003 Leu Leu Asn 250gtggttctcc accttctagt cccactctag aaaccacatt agacagaagg tctcctgcta 1063tggtgctttc cccatcccta atctcttaga ttttcctcaa gactcccttc tcagagaaca 1123cgctctgtcc atgtccccag ctggcttctc agcctagcct ttgagggccc tgtggggagg 1183cggggacaag aaagcagaaa agtcttggcc ccgagccagt ggttaggtcc taggaattgg 1243ctggagtgga ggccagaaag cctgggcaga tgatgagagc ccagctgggc tgtcactgca 1303ggttccgggg cctacagccc tgggtcagca gagtatgagt tcccagactt tccagaaggt 1363ccttagcaat gtcccagaaa ttcaccgtac acttctcagt gtcttaggag ggcccgggat 1423ccagatgtct ggttcatccc tgaatcctct ccctccttct tgctcgtatg gtgggagtgg 1483tggccagggg aagatgagtg gtgtcccgga tgatgcctgt caaggtccca cctcccctcc 1543ggctgttctc atgacagctg tttggttctc catgacccct atctagatgt agaggcatgg 1603agtgagtcag ggatttcccg aacttgagtt ttaccactcc tcctagtggc tgccttaggg 1663gaatgggaag aacccagtgt gggggcaccc attagaatct ttgcccggct cctcacaatg 1723ccctagggtc ccctagggta cccgctccct ctgtttagtc tacccagaga tgctcctgag 1783ctcacctaga gggtagggac ggtaggctcc aggtccaacc tctccaggtc agcaccctgc 1843catgctgctg ctcctcatta acaaacctgc ttgtctcctc ctgcgcccct tctcagtcag 1903ccagggtctg aggggaaggg cctcccgttt ccccatccgt cagacatggt tgactgcttt 1963gcattttggg ctcttctatc tattttgtaa aataagacat cagatccaat aaaacacacg 2023gctatgcaca aaaaaaaaaa aaaaaaaaaa aaaa 2057 42 252 PRT Rattus sp. 42 MetArg Gly Gln Gly Arg Lys Glu Ser Leu Ser Glu Ser Arg Asp Leu 1 5 10 15Asp Gly Ser Tyr Asp Gln Leu Thr Gly His Pro Pro Gly Pro Ser Lys 20 25 30Lys Ala Leu Lys Gln Arg Phe Leu Lys Leu Leu Pro Cys Cys Gly Pro 35 40 45Gln Ala Leu Pro Ser Val Ser Glu Asn Ser Val Glu Asp Glu Phe Glu 50 55 60Leu Ser Thr Val Cys His Arg Pro Glu Gly Leu Glu Gln Leu Gln Glu 65 70 7580 Gln Thr Lys Phe Thr Arg Arg Glu Leu Gln Val Leu Tyr Arg Gly Phe 85 9095 Lys Asn Glu Cys Pro Ser Gly Ile Val Asn Glu Glu Asn Phe Lys Gln 100105 110 Ile Tyr Ser Gln Phe Phe Pro Gln Gly Asp Ser Ser Asn Tyr Ala Thr115 120 125 Phe Leu Phe Asn Ala Phe Asp Thr Asn His Asp Gly Ser Val SerPhe 130 135 140 Glu Asp Phe Val Ala Gly Leu Ser Val Ile Leu Arg Gly ThrIle Asp 145 150 155 160 Asp Arg Leu Ser Trp Ala Phe Asn Leu Tyr Asp LeuAsn Lys Asp Gly 165 170 175 Cys Ile Thr Lys Glu Glu Met Leu Asp Ile MetLys Ser Ile Tyr Asp 180 185 190 Met Met Gly Lys Tyr Thr Tyr Pro Ala LeuArg Glu Glu Ala Pro Arg 195 200 205 Glu His Val Glu Ser Phe Phe Gln LysMet Asp Arg Asn Lys Asp Gly 210 215 220 Val Val Thr Ile Glu Glu Phe IleGlu Ser Cys Gln Gln Asp Glu Asn 225 230 235 240 Ile Met Arg Ser Met GlnLeu Ser Pro Leu Leu Asn 245 250 43 26 PRT Artificial Sequence Xaas atpositions 2,5,6,9,17,25 and 26 may be Ile, Leu, Val or Met 43 Glu XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Asp Lys Asp Gly Asp Gly Xaa 1 5 10 15 XaaXaa Xaa Xaa Glu Phe Xaa Xaa Xaa Xaa 20 25 44 40 DNA Rattus sp. 44taatacgact cactataggg actggccatc ctgctctcag 40 45 40 DNA Rattus sp. 45attaaccctc actaaaggga cactactgtt taagctcaag 40 46 40 DNA Rattus sp. 46taatacgact cactataggg cacctcccct ccggctgttc 40 47 40 DNA Rattus sp. 47attaaccctc actaaaggga gagcagcagc atggcagggt 40 48 2413 DNA Simian sp.CDS (265)..(963) 48 gtcgacccac gcgtccggtg cgctgtggtt gcgggggggagccccgccag ccaaatgcca 60 ggatcagcat gagaggctgg actttagtcc aggtctgtcctcaccccggg ggaccgccgg 120 ctttgcaggg tgcagctgcg aggaactgct cacttttttccccttgcaag tctttgttcc 180 aagcctgacg ttgctacgat tctgtaatta actccctccactccaaaggg gtctggaggc 240 tgggatgctc tgccagctca gagg atg ttg act ctg gagtgg gag tcc gaa 291 Met Leu Thr Leu Glu Trp Glu Ser Glu 1 5 gga ctg caaaca gtg ggt att gtt gtg att ata tgt gca tct ctg aag 339 Gly Leu Gln ThrVal Gly Ile Val Val Ile Ile Cys Ala Ser Leu Lys 10 15 20 25 ctg ctt catttg ctg gga ctg att gat ttt tcg gaa gac agc gtg gaa 387 Leu Leu His LeuLeu Gly Leu Ile Asp Phe Ser Glu Asp Ser Val Glu 30 35 40 gat gaa ctg gagatg gcc act gtc agg cat cgg cct gag gcc ctt gag 435 Asp Glu Leu Glu MetAla Thr Val Arg His Arg Pro Glu Ala Leu Glu 45 50 55 ctt ctg gaa gcc cagagc aaa ttt acc aag aaa gag ctt cag atc ctt 483 Leu Leu Glu Ala Gln SerLys Phe Thr Lys Lys Glu Leu Gln Ile Leu 60 65 70 tac aga gga ttt aag aacgaa tgc ccc agt ggt gtt gtt aat gaa gaa 531 Tyr Arg Gly Phe Lys Asn GluCys Pro Ser Gly Val Val Asn Glu Glu 75 80 85 acc ttc aaa gag att tac tcgcag ttc ttt cca cag gga gac tct aca 579 Thr Phe Lys Glu Ile Tyr Ser GlnPhe Phe Pro Gln Gly Asp Ser Thr 90 95 100 105 aca tat gca cat ttt ctgttc aat gcg ttt gat acg gac cac aat gga 627 Thr Tyr Ala His Phe Leu PheAsn Ala Phe Asp Thr Asp His Asn Gly 110 115 120 gct gtg agt ttc gag gatttc atc aaa ggt ctt tcc att ttg ctc cgg 675 Ala Val Ser Phe Glu Asp PheIle Lys Gly Leu Ser Ile Leu Leu Arg 125 130 135 ggg aca gta caa gaa aaactc aat tgg gca ttt aat ctg tat gat ata 723 Gly Thr Val Gln Glu Lys LeuAsn Trp Ala Phe Asn Leu Tyr Asp Ile 140 145 150 aat aaa gat ggc tac atcact aaa gag gaa atg ctt gat ata atg aaa 771 Asn Lys Asp Gly Tyr Ile ThrLys Glu Glu Met Leu Asp Ile Met Lys 155 160 165 gca ata tac gac atg atgggt aaa tgt aca tat cct gtc ctc aaa gaa 819 Ala Ile Tyr Asp Met Met GlyLys Cys Thr Tyr Pro Val Leu Lys Glu 170 175 180 185 gat gca ccc aga caacac gtc gaa aca ttt ttt cag aaa atg gac aaa 867 Asp Ala Pro Arg Gln HisVal Glu Thr Phe Phe Gln Lys Met Asp Lys 190 195 200 aat aaa gat ggg gttgtt acc ata gat gag ttc att gaa agc tgc caa 915 Asn Lys Asp Gly Val ValThr Ile Asp Glu Phe Ile Glu Ser Cys Gln 205 210 215 aaa gat gaa aac ataatg cgc tcc atg cag ctc ttt gaa aat gtg att 963 Lys Asp Glu Asn Ile MetArg Ser Met Gln Leu Phe Glu Asn Val Ile 220 225 230 taacttgtcaactagatcct gaatccaaca gacaaatgtg aactattcta ccacccttaa 1023 agtcggagctaccactttta gcatagattg ctcagcttga cactgaagca tattatgcaa 1083 acaagctttgttttaatata aagcaatccc caaaagattt gagtttctca gttataaatt 1143 tgcatcctttccataatgcc actgagttca tgggatgttc taactcattt catactctgt 1203 gaatattcaaaagtaataga atctggcata tagttttatt gattccttag ccatgggatt 1263 attgaggctttcacatatca gtgattttaa aataccagtg ttttttgctc tcatttgtat 1323 gtattcagtcctaggatttt gaatggtttt ctaatatact gacatctgca tttaatttcc 1383 agaaattaaattaattttca tgtctgaatg ctgtaattcc atttatatac tttaagtaaa 1443 caaataagattactacaatt aaacacatag ttccagtttc tatggccttc ccttcccacc 1503 ttctattataaattaatttt atctggtatt tttaaacatt taaaaattta tcatcagata 1563 tcagcatatgcctaattatg cctaatgaaa cttaataagc atttaatttt ccatcataca 1623 ttatagccaaggcctatata ctatatataa ttttggattt gtttaatctt acaggctgtt 1683 ttccattgtatcatcaagtg gaagttcaag acggcatcaa acaaaacaag gatgtttaca 1743 gacatatgcaaagggtcagg atatctatcc tccagtatat gttaatgctt aataacaagt 1803 aatcctaacagcattaaagg ccaaatctgt cctctttccc ctgacttcct tacagcatgt 1863 ttatattacaagccattcag ggacaaagaa accttgacta ccccactgtc tactaggaac 1923 aaacaaacagcaagcaaaat tcactttgaa agcaccagtg gttccattac attgacaact 1983 actaccaagattcagtagaa aataagtgct caacaactaa tccagattac aatatgattt 2043 agtgcatcataaaattccaa caattcagat tatttttaat catctcagcc acaactgtaa 2103 agttgccacattactaaaga cacacacatc gtccctgttt tgtagaaata tcacaaagac 2163 caagaggctacagaaggagg aaatttgcaa ctgtctttgc aacaataaat caggtatcta 2223 ttctggtgtagagataggat gttgaaagct gccctgctat caccagtgta gaaattaaga 2283 gtagtacaatacatgtacac tgaaatttgc catcgcgtgt ttgtgtaaac tcaatgtgca 2343 cattttgtatttcaaaaaga aaaaataaaa gcaaaataaa atgttwawaa mwmwaaaaaa 2403 aaaaaaaaaa2413 49 233 PRT Simian sp. 49 Met Leu Thr Leu Glu Trp Glu Ser Glu GlyLeu Gln Thr Val Gly Ile 1 5 10 15 Val Val Ile Ile Cys Ala Ser Leu LysLeu Leu His Leu Leu Gly Leu 20 25 30 Ile Asp Phe Ser Glu Asp Ser Val GluAsp Glu Leu Glu Met Ala Thr 35 40 45 Val Arg His Arg Pro Glu Ala Leu GluLeu Leu Glu Ala Gln Ser Lys 50 55 60 Phe Thr Lys Lys Glu Leu Gln Ile LeuTyr Arg Gly Phe Lys Asn Glu 65 70 75 80 Cys Pro Ser Gly Val Val Asn GluGlu Thr Phe Lys Glu Ile Tyr Ser 85 90 95 Gln Phe Phe Pro Gln Gly Asp SerThr Thr Tyr Ala His Phe Leu Phe 100 105 110 Asn Ala Phe Asp Thr Asp HisAsn Gly Ala Val Ser Phe Glu Asp Phe 115 120 125 Ile Lys Gly Leu Ser IleLeu Leu Arg Gly Thr Val Gln Glu Lys Leu 130 135 140 Asn Trp Ala Phe AsnLeu Tyr Asp Ile Asn Lys Asp Gly Tyr Ile Thr 145 150 155 160 Lys Glu GluMet Leu Asp Ile Met Lys Ala Ile Tyr Asp Met Met Gly 165 170 175 Lys CysThr Tyr Pro Val Leu Lys Glu Asp Ala Pro Arg Gln His Val 180 185 190 GluThr Phe Phe Gln Lys Met Asp Lys Asn Lys Asp Gly Val Val Thr 195 200 205Ile Asp Glu Phe Ile Glu Ser Cys Gln Lys Asp Glu Asn Ile Met Arg 210 215220 Ser Met Gln Leu Phe Glu Asn Val Ile 225 230 50 1591 DNA Simian sp.CDS (265)..(963) 50 gtcgacccac gcgtccggtg cgctgtggtt gcgggggggagccccgccag ccaaatgcca 60 ggatcagcat gagaggctgg actttagtcc aggtctgtcctcaccccggg ggaccgccgg 120 ctttgcaggg tgcagctgcg aggaactgct cacttttttccccttgcaag tctttgttcc 180 aagcctgacg ttgctacgat tctgtaatta actccctccactccaaaggg gtctggaggc 240 tgggatgctc tgccagctca gagg atg ttg act ctg gagtgg gag tcc gaa 291 Met Leu Thr Leu Glu Trp Glu Ser Glu 1 5 gga ctg caaaca gtg ggt att gtt gtg att ata tgt gca tct ctg aag 339 Gly Leu Gln ThrVal Gly Ile Val Val Ile Ile Cys Ala Ser Leu Lys 10 15 20 25 ctg ctt catttg ctg gga ctg att gat ttt tcg gaa gac agc gtg gaa 387 Leu Leu His LeuLeu Gly Leu Ile Asp Phe Ser Glu Asp Ser Val Glu 30 35 40 gat gaa ctg gagatg gcc act gtc agg cat cgg cct gag gcc ctt gag 435 Asp Glu Leu Glu MetAla Thr Val Arg His Arg Pro Glu Ala Leu Glu 45 50 55 ctt ctg gaa gcc cagagc aaa ttt acc aag aaa gag ctt cag atc ctt 483 Leu Leu Glu Ala Gln SerLys Phe Thr Lys Lys Glu Leu Gln Ile Leu 60 65 70 tac aga gga ttt aag aacgaa tgc ccc agt ggt gtt gtt aat gaa gaa 531 Tyr Arg Gly Phe Lys Asn GluCys Pro Ser Gly Val Val Asn Glu Glu 75 80 85 acc ttc aaa gag att tac tcgcag ttc ttt cca cag gga gac tct aca 579 Thr Phe Lys Glu Ile Tyr Ser GlnPhe Phe Pro Gln Gly Asp Ser Thr 90 95 100 105 aca tat gca cat ttt ctgttc aat gcg ttt gat acg gac cac aat gga 627 Thr Tyr Ala His Phe Leu PheAsn Ala Phe Asp Thr Asp His Asn Gly 110 115 120 gct gtg agt ttc gag gatttc atc aaa ggt ctt tcc att ttg ctc cgg 675 Ala Val Ser Phe Glu Asp PheIle Lys Gly Leu Ser Ile Leu Leu Arg 125 130 135 ggg aca gta caa gaa aaactc aat tgg gca ttt aat ctg tat gat ata 723 Gly Thr Val Gln Glu Lys LeuAsn Trp Ala Phe Asn Leu Tyr Asp Ile 140 145 150 aat aaa gat ggc tac atcact aaa gag gaa atg ctt gat ata atg aaa 771 Asn Lys Asp Gly Tyr Ile ThrLys Glu Glu Met Leu Asp Ile Met Lys 155 160 165 gca ata tac gac atg atgggt aaa tgt aca tat cct gtc ctc aaa gaa 819 Ala Ile Tyr Asp Met Met GlyLys Cys Thr Tyr Pro Val Leu Lys Glu 170 175 180 185 gat gca ccc aga caacac gtc gaa aca ttt ttt cag gct gtt ttc cat 867 Asp Ala Pro Arg Gln HisVal Glu Thr Phe Phe Gln Ala Val Phe His 190 195 200 tgt atc atc aag tggaag ttc aag acg gca tca aac aaa aca agg atg 915 Cys Ile Ile Lys Trp LysPhe Lys Thr Ala Ser Asn Lys Thr Arg Met 205 210 215 ttt aca gac ata tgcaaa ggg tca gga tat cta tcc tcc agt ata tgt 963 Phe Thr Asp Ile Cys LysGly Ser Gly Tyr Leu Ser Ser Ser Ile Cys 220 225 230 taatgcttaataacaagtaa tcctaacagc attaaaggcc aaatctgtcc tctttcccct 1023 gacttccttacagcatgttt atattacaag ccattcaggg acaaagaaac cttgactacc 1083 ccactgtctactaggaacaa acaaacagca agcaaaattc actttgaaag caccagtggt 1143 tccattacattgacaactac taccaagatt cagtagaaaa taagtgctca acaactaatc 1203 cagattacaatatgatttag tgcatcataa aattccaaca attcagatta tttttaatca 1263 tctcagccacaactgtaaag ttgccacatt actaaagaca cacacatcgt ccctgttttg 1323 tagaaatatcacaaagacca agaggctaca gaaggaggaa atttgcaact gtctttgcaa 1383 caataaatcaggtatctatt ctggtgtaga gataggatgt tgaaagctgc cctgctatca 1443 ccagtgtagaaattaagagt agtacaatac atgtacactg aaatttgcca tcgcgtgttt 1503 gtgtaaactcaatgtgcaca ttttgtattt caaaaagaaa aaataaaagc aaaataaaat 1563 gttwawaamwmwaaaaaaaa aaaaaaaa 1591 51 233 PRT Simian sp. 51 Met Leu Thr Leu GluTrp Glu Ser Glu Gly Leu Gln Thr Val Gly Ile 1 5 10 15 Val Val Ile IleCys Ala Ser Leu Lys Leu Leu His Leu Leu Gly Leu 20 25 30 Ile Asp Phe SerGlu Asp Ser Val Glu Asp Glu Leu Glu Met Ala Thr 35 40 45 Val Arg His ArgPro Glu Ala Leu Glu Leu Leu Glu Ala Gln Ser Lys 50 55 60 Phe Thr Lys LysGlu Leu Gln Ile Leu Tyr Arg Gly Phe Lys Asn Glu 65 70 75 80 Cys Pro SerGly Val Val Asn Glu Glu Thr Phe Lys Glu Ile Tyr Ser 85 90 95 Gln Phe PhePro Gln Gly Asp Ser Thr Thr Tyr Ala His Phe Leu Phe 100 105 110 Asn AlaPhe Asp Thr Asp His Asn Gly Ala Val Ser Phe Glu Asp Phe 115 120 125 IleLys Gly Leu Ser Ile Leu Leu Arg Gly Thr Val Gln Glu Lys Leu 130 135 140Asn Trp Ala Phe Asn Leu Tyr Asp Ile Asn Lys Asp Gly Tyr Ile Thr 145 150155 160 Lys Glu Glu Met Leu Asp Ile Met Lys Ala Ile Tyr Asp Met Met Gly165 170 175 Lys Cys Thr Tyr Pro Val Leu Lys Glu Asp Ala Pro Arg Gln HisVal 180 185 190 Glu Thr Phe Phe Gln Ala Val Phe His Cys Ile Ile Lys TrpLys Phe 195 200 205 Lys Thr Ala Ser Asn Lys Thr Arg Met Phe Thr Asp IleCys Lys Gly 210 215 220 Ser Gly Tyr Leu Ser Ser Ser Ile Cys 225 230 522051 DNA Rattus sp. CDS (85)..(1305) 52 ggtggagcta agcactcact gcggtgctgccctgcgtctg cagagaacaa ggaaagcttc 60 tctgcagggc tgtcagctgc caaa atg aacggc gtg gaa ggg aac aac gag 111 Met Asn Gly Val Glu Gly Asn Asn Glu 1 5ctc cct ctc gct aac acc tcg acc tcc gcc ctt gtc ccg gaa gat ctg 159 LeuPro Leu Ala Asn Thr Ser Thr Ser Ala Leu Val Pro Glu Asp Leu 10 15 20 25gat ctg aag caa gac cag ccg ctc agc gag gaa act gac acg gtg cgg 207 AspLeu Lys Gln Asp Gln Pro Leu Ser Glu Glu Thr Asp Thr Val Arg 30 35 40 gagatg gag gct gca ggt gag gcc ggt gcg gag gga ggc gcg tcc ccc 255 Glu MetGlu Ala Ala Gly Glu Ala Gly Ala Glu Gly Gly Ala Ser Pro 45 50 55 gat tcggag cac tgc gac ccc cag ctc tgc ctc cga gtg gct gag aat 303 Asp Ser GluHis Cys Asp Pro Gln Leu Cys Leu Arg Val Ala Glu Asn 60 65 70 ggc tgt gctgcc gca gcg gga gag ggg ctg gag gat ggt ctg tct tca 351 Gly Cys Ala AlaAla Ala Gly Glu Gly Leu Glu Asp Gly Leu Ser Ser 75 80 85 tca aag tgt ggggac gca ccc ttg gcg tct gtg gca gcc aac gac agc 399 Ser Lys Cys Gly AspAla Pro Leu Ala Ser Val Ala Ala Asn Asp Ser 90 95 100 105 aat aaa aatggc tgt cag ctt gca ggg ccg ctc agc cct gct aag cca 447 Asn Lys Asn GlyCys Gln Leu Ala Gly Pro Leu Ser Pro Ala Lys Pro 110 115 120 aaa act ctggaa gcc agt ggt gca gtg ggc ctg ggg tcg cag atg atg 495 Lys Thr Leu GluAla Ser Gly Ala Val Gly Leu Gly Ser Gln Met Met 125 130 135 cca ggg ccgaag aag acc aag gta atg act acc aag ggc gcc atc tct 543 Pro Gly Pro LysLys Thr Lys Val Met Thr Thr Lys Gly Ala Ile Ser 140 145 150 gcg act acaggc aag gaa gga gaa gca ggg gcg gca atg cag gaa aag 591 Ala Thr Thr GlyLys Glu Gly Glu Ala Gly Ala Ala Met Gln Glu Lys 155 160 165 aag ggg gtgcag aaa gaa aaa aag gca gct gga gga ggg aaa gac gag 639 Lys Gly Val GlnLys Glu Lys Lys Ala Ala Gly Gly Gly Lys Asp Glu 170 175 180 185 act cgtcct aga gcc cct aag atc aat aac tgc atg gac tcc ctg gaa 687 Thr Arg ProArg Ala Pro Lys Ile Asn Asn Cys Met Asp Ser Leu Glu 190 195 200 gcc atcgat caa gag ctg tca aat gta aat gcg caa gct gac agg gcc 735 Ala Ile AspGln Glu Leu Ser Asn Val Asn Ala Gln Ala Asp Arg Ala 205 210 215 ttc ctccag ctg gaa cgc aaa ttt ggg cgg atg aga agg ctc cac atg 783 Phe Leu GlnLeu Glu Arg Lys Phe Gly Arg Met Arg Arg Leu His Met 220 225 230 cag cgccga agt ttc atc atc caa aac atc cca ggt ttc tgg gtc aca 831 Gln Arg ArgSer Phe Ile Ile Gln Asn Ile Pro Gly Phe Trp Val Thr 235 240 245 gcg tttcgg aac cac ccg caa ctg tca ccg atg atc agt ggc caa gat 879 Ala Phe ArgAsn His Pro Gln Leu Ser Pro Met Ile Ser Gly Gln Asp 250 255 260 265 gaagac atg atg agg tac atg atc aat tta gag gtg gag gag ctt aag 927 Glu AspMet Met Arg Tyr Met Ile Asn Leu Glu Val Glu Glu Leu Lys 270 275 280 caccca aga gca ggg tgc aaa ttt aag ttc atc ttc caa agc aac ccc 975 His ProArg Ala Gly Cys Lys Phe Lys Phe Ile Phe Gln Ser Asn Pro 285 290 295 tacttc cga aat gag ggg ctg gtc aaa gag tac gag cgc aga tcc tca 1023 Tyr PheArg Asn Glu Gly Leu Val Lys Glu Tyr Glu Arg Arg Ser Ser 300 305 310 ggtcga gtg gtg tcg ctc tct acg cca atc cgc tgg cac cgg ggt caa 1071 Gly ArgVal Val Ser Leu Ser Thr Pro Ile Arg Trp His Arg Gly Gln 315 320 325 gaaccc cag gcc cat atc cac agg aat aga gag ggg aac acg att ccc 1119 Glu ProGln Ala His Ile His Arg Asn Arg Glu Gly Asn Thr Ile Pro 330 335 340 345agt ttc ttc aat tgg ttc tca gac cac agc ctc cta gaa ttc gac aga 1167 SerPhe Phe Asn Trp Phe Ser Asp His Ser Leu Leu Glu Phe Asp Arg 350 355 360ata gct gaa att atc aaa ggg gag ctt tgg tcc aat ccc cta caa tac 1215 IleAla Glu Ile Ile Lys Gly Glu Leu Trp Ser Asn Pro Leu Gln Tyr 365 370 375tac ctg atg ggc gat ggg cca cgc aga gga gtt cga gtc cca cca agg 1263 TyrLeu Met Gly Asp Gly Pro Arg Arg Gly Val Arg Val Pro Pro Arg 380 385 390cag cca gtg gag agt ccc agg tcc ttc agg ttc cag tct ggc 1305 Gln Pro ValGlu Ser Pro Arg Ser Phe Arg Phe Gln Ser Gly 395 400 405 taagctctgccctcgtgaga agctcttaca gaagagtcct taccaccttc tcagcttggc 1365 tagcagcatgcagccttctg tctgctttct cttccttgga ttgtgtcctt tggttcttct 1425 aagtctccggtagtttcaag gttgtggctt ccaagtcttt gctcttcttt ctcttggcca 1485 tcacgatgtcctgcatagtg ttaatggtgt tccaagtgca tggcctccaa actgcttcta 1545 tgccaagctcacgtgctgta gtttgtactg cttttctttg catggcttgg ttcctgtctg 1605 tgatcttctaggttttttgt tttctttttt aaaagtggtt ctctatcaaa agaaagcttg 1665 acatatccttaccaagaact agccagattt catactgtgt tcccgatatc tatgtactgt 1725 gaagaactgtgagtttcgcc actgcaagat gggactgtat cccaatccag ccatcagccc 1785 aacaggacattccaagctgt caccaactga tcctagctgt cttcctgggc ctttgccatt 1845 taccctgctttttatctata gaatgagcag gtggctggta ggtgactact aggtaagagt 1905 gaagtattaggtgaggagtg ttttctgtca ccacattgtt cttgtaccaa tgcatcatga 1965 tcagcttggatcagctactg actgtctgat atttctaacc cccaacacaa aaaaaaaaaa 2025 aaaaaaaaaaaaaaaaaaaa aaaaaa 2051 53 407 PRT Rattus sp. 53 Met Asn Gly Val Glu GlyAsn Asn Glu Leu Pro Leu Ala Asn Thr Ser 1 5 10 15 Thr Ser Ala Leu ValPro Glu Asp Leu Asp Leu Lys Gln Asp Gln Pro 20 25 30 Leu Ser Glu Glu ThrAsp Thr Val Arg Glu Met Glu Ala Ala Gly Glu 35 40 45 Ala Gly Ala Glu GlyGly Ala Ser Pro Asp Ser Glu His Cys Asp Pro 50 55 60 Gln Leu Cys Leu ArgVal Ala Glu Asn Gly Cys Ala Ala Ala Ala Gly 65 70 75 80 Glu Gly Leu GluAsp Gly Leu Ser Ser Ser Lys Cys Gly Asp Ala Pro 85 90 95 Leu Ala Ser ValAla Ala Asn Asp Ser Asn Lys Asn Gly Cys Gln Leu 100 105 110 Ala Gly ProLeu Ser Pro Ala Lys Pro Lys Thr Leu Glu Ala Ser Gly 115 120 125 Ala ValGly Leu Gly Ser Gln Met Met Pro Gly Pro Lys Lys Thr Lys 130 135 140 ValMet Thr Thr Lys Gly Ala Ile Ser Ala Thr Thr Gly Lys Glu Gly 145 150 155160 Glu Ala Gly Ala Ala Met Gln Glu Lys Lys Gly Val Gln Lys Glu Lys 165170 175 Lys Ala Ala Gly Gly Gly Lys Asp Glu Thr Arg Pro Arg Ala Pro Lys180 185 190 Ile Asn Asn Cys Met Asp Ser Leu Glu Ala Ile Asp Gln Glu LeuSer 195 200 205 Asn Val Asn Ala Gln Ala Asp Arg Ala Phe Leu Gln Leu GluArg Lys 210 215 220 Phe Gly Arg Met Arg Arg Leu His Met Gln Arg Arg SerPhe Ile Ile 225 230 235 240 Gln Asn Ile Pro Gly Phe Trp Val Thr Ala PheArg Asn His Pro Gln 245 250 255 Leu Ser Pro Met Ile Ser Gly Gln Asp GluAsp Met Met Arg Tyr Met 260 265 270 Ile Asn Leu Glu Val Glu Glu Leu LysHis Pro Arg Ala Gly Cys Lys 275 280 285 Phe Lys Phe Ile Phe Gln Ser AsnPro Tyr Phe Arg Asn Glu Gly Leu 290 295 300 Val Lys Glu Tyr Glu Arg ArgSer Ser Gly Arg Val Val Ser Leu Ser 305 310 315 320 Thr Pro Ile Arg TrpHis Arg Gly Gln Glu Pro Gln Ala His Ile His 325 330 335 Arg Asn Arg GluGly Asn Thr Ile Pro Ser Phe Phe Asn Trp Phe Ser 340 345 350 Asp His SerLeu Leu Glu Phe Asp Arg Ile Ala Glu Ile Ile Lys Gly 355 360 365 Glu LeuTrp Ser Asn Pro Leu Gln Tyr Tyr Leu Met Gly Asp Gly Pro 370 375 380 ArgArg Gly Val Arg Val Pro Pro Arg Gln Pro Val Glu Ser Pro Arg 385 390 395400 Ser Phe Arg Phe Gln Ser Gly 405 54 4148 DNA Homo sapiens CDS(88)..(1329) 54 ggggtggtgc tagacgtttc gggcagagct cggccgctgc ggaggacaaggaactctccc 60 tctcccacta gtctgacttc ttccaaa atg agc ggc ctg gat ggg ggcaac aag 114 Met Ser Gly Leu Asp Gly Gly Asn Lys 1 5 ctc cct ctc gcc caaacc ggc ggc ctg gct gct ccc gac cat gcc tca 162 Leu Pro Leu Ala Gln ThrGly Gly Leu Ala Ala Pro Asp His Ala Ser 10 15 20 25 gga gat ccg gac ctagac cag tgc caa ggg ctc cgt gaa gaa acc gag 210 Gly Asp Pro Asp Leu AspGln Cys Gln Gly Leu Arg Glu Glu Thr Glu 30 35 40 gcg aca cag gtg atg gcgaac aca ggt ggg ggc agc ctg gag acc gtt 258 Ala Thr Gln Val Met Ala AsnThr Gly Gly Gly Ser Leu Glu Thr Val 45 50 55 gcg gag ggg ggt gca tcc caggat cct gtc gac tgt ggc ccc gcg ctc 306 Ala Glu Gly Gly Ala Ser Gln AspPro Val Asp Cys Gly Pro Ala Leu 60 65 70 cgc gtc cca gtt gcc ggg agt cgcggc ggt gca gcg acc aaa gcc ggg 354 Arg Val Pro Val Ala Gly Ser Arg GlyGly Ala Ala Thr Lys Ala Gly 75 80 85 cag gag gat gct cca cct tct acg aaaggt ctg gaa gca gcc tct gcc 402 Gln Glu Asp Ala Pro Pro Ser Thr Lys GlyLeu Glu Ala Ala Ser Ala 90 95 100 105 gcc gag gct gct gac agc agc cagaaa aat ggc tgt cag ctt gga gag 450 Ala Glu Ala Ala Asp Ser Ser Gln LysAsn Gly Cys Gln Leu Gly Glu 110 115 120 ccc cgt ggc cct gct ggg cag aaggct cta gaa gcc tgt ggc gca ggg 498 Pro Arg Gly Pro Ala Gly Gln Lys AlaLeu Glu Ala Cys Gly Ala Gly 125 130 135 ggc ttg ggg tct cag atg ata ccgggg aag aag gcc aag gaa gtg acg 546 Gly Leu Gly Ser Gln Met Ile Pro GlyLys Lys Ala Lys Glu Val Thr 140 145 150 act aaa aaa cgc gcc atc tcg gcagca gtg gaa aag gag gga gaa gca 594 Thr Lys Lys Arg Ala Ile Ser Ala AlaVal Glu Lys Glu Gly Glu Ala 155 160 165 ggg gcg gcg atg gag gaa aag aaggta gtg cag aag gaa aaa aag gtg 642 Gly Ala Ala Met Glu Glu Lys Lys ValVal Gln Lys Glu Lys Lys Val 170 175 180 185 gca gga ggg gtg aaa gag gagaca cgg ccc agg gcc ccg aag atc aat 690 Ala Gly Gly Val Lys Glu Glu ThrArg Pro Arg Ala Pro Lys Ile Asn 190 195 200 aac tgc atg gac tca ctg gaggcc atc gat caa gag ttg tca aac gta 738 Asn Cys Met Asp Ser Leu Glu AlaIle Asp Gln Glu Leu Ser Asn Val 205 210 215 aat gcc cag gct gac agg gccttc ctt cag ctt gag cgc aag ttt ggc 786 Asn Ala Gln Ala Asp Arg Ala PheLeu Gln Leu Glu Arg Lys Phe Gly 220 225 230 cgc atg cga agg ctc cac atgcag cgc aga agt ttc att atc cag aat 834 Arg Met Arg Arg Leu His Met GlnArg Arg Ser Phe Ile Ile Gln Asn 235 240 245 atc cca ggt ttc tgg gtt actgcc ttt cga aac cac ccc cag ctg tca 882 Ile Pro Gly Phe Trp Val Thr AlaPhe Arg Asn His Pro Gln Leu Ser 250 255 260 265 cct atg atc agt ggc caagat gaa gac atg ctg agg tac atg atc aat 930 Pro Met Ile Ser Gly Gln AspGlu Asp Met Leu Arg Tyr Met Ile Asn 270 275 280 ttg gag gtg gag gag cttaaa cac ccc aga gca ggc tgc aaa ttc aag 978 Leu Glu Val Glu Glu Leu LysHis Pro Arg Ala Gly Cys Lys Phe Lys 285 290 295 ttc atc ttt cag ggc aacccc tac ttc cga aat gag ggg ctt gtc aag 1026 Phe Ile Phe Gln Gly Asn ProTyr Phe Arg Asn Glu Gly Leu Val Lys 300 305 310 gaa tat gaa cgc aga tcctct ggc cgg gtg gtg tct ctt tcc act cca 1074 Glu Tyr Glu Arg Arg Ser SerGly Arg Val Val Ser Leu Ser Thr Pro 315 320 325 atc cgc tgg cac cga ggccaa gac ccc cag gct cat atc cac aga aac 1122 Ile Arg Trp His Arg Gly GlnAsp Pro Gln Ala His Ile His Arg Asn 330 335 340 345 cgg gaa ggg aac actatc cct agt ttc ttc aac tgg ttt tca gac cac 1170 Arg Glu Gly Asn Thr IlePro Ser Phe Phe Asn Trp Phe Ser Asp His 350 355 360 agc ctt cta gaa ttcgac aga att gca gag att atc aaa gga gaa ctg 1218 Ser Leu Leu Glu Phe AspArg Ile Ala Glu Ile Ile Lys Gly Glu Leu 365 370 375 tgg ccc aat ccc ctacaa tac tac ctg atg ggt gaa ggg ccc cgt aga 1266 Trp Pro Asn Pro Leu GlnTyr Tyr Leu Met Gly Glu Gly Pro Arg Arg 380 385 390 gga att cga ggc ccacca agg cag cca gtg gag agc gcc aga tcc ttc 1314 Gly Ile Arg Gly Pro ProArg Gln Pro Val Glu Ser Ala Arg Ser Phe 395 400 405 agg ttc cag tct ggctaatctctgt cctgtgagaa gcttctgcac aagtttcctt 1369 Arg Phe Gln Ser Gly 410accacctcct cttggaccta tgcttggcca acagcatgca gtcttccatc tgctttctct 1429tcatactgtg gattatcttt tcctttggtt ctaaatcttc agtaatcggt tgcaagattg 1489ttggcttacc tgcctgtgcc attcttcctc tgggccttca tgcttttctg cattgtgtta 1549acatgtttca agtgcatggc cttctacggc ttctatgcca agcgtatgat actatagata 1609tagtgtacca tactgccttt ctttgcatgg cttggaccct atctgtgacc atgctcttct 1669cccaatttaa gtggttctgt accacaaaga atcttgatac attttcacaa ataactgatt 1729gggcttcata ctttatgctg gctgtgtcct gatacccatg tacttatggt aagctatttg 1789ggtattacca ctgcaagaca aaactgatat cttaacccgg ccatcaaccc aaattggaca 1849ttccagacta ccaccaactg gatcccagct gccttcctgg gcttgtgcca tccaccctac 1909tggttatctg atagaacaag ctggtggctg atgggtgact gctaggcgtg actgaggtaa 1969tagatgaaaa gtgttctatg ttatcacatt ggttttcctg tacctttggt tactctacgt 2029catgaccagc tgctggtgag tatgaagcct gtgctatagc ccacccctac tcactctcac 2089cttctggttg aactttgctt aggccaccat tgtctgcctc atcaggaact atctgtagac 2149gtagctccca gggagctcac agcaacaccc cctaccacca ggatgggcag taatatgtga 2209cagagcccaa agcaaggctg gaacgcagtc ccttccagct tagtctttct gactcctagc 2269caacaaacca tccttaatgt gagcaacttc tttaggcatt tcctcttttc cccgcctgca 2329cccactctga acatgacaaa agttgccaga gttggggcat tgaggaagag atatttctgg 2389aatgtgagac ttgttatgcc tctgtctctt tctctccctc cccctcccct ctccctcccc 2449ctctccctcc catccctttt cttccctttc actctgaagc agttttagct tattaacaga 2509aaacaaaact ggcaaagcag gctttttgtt taatttgctc tttccctgat tgtgttcaga 2569gagaaaggtt atgattaaat gggctccaga tctcttattg cccttattcc tccaccccac 2629ttcttttagc aaggtctgaa agtttcaaag ggagacctat aggttaattg tttagttata 2689ggcagtgtta aattaggcag attttgacat atttatcttt ttaccccatc cattctacca 2749aaacctgtgt atttcttgag tttttagttt gagaagctgg aaagagagag aagggcctca 2809cagtgatggg ttcaggacgg gtcaaaggca aaggcctttg tgatgtgagc aaaggcaacc 2869aaaacttagc ctcactccac ttttctaaag atggaaattc ttttttgggc cttggactgc 2929ttctagggta gcattttgta ggtcactctt ctcctttgta ctattttgtt tctgccctga 2989tgtcccttgg gtctccatcc tactgcctgg ctttcttggc cctcatttct cagcttctgc 3049atttccttcc ctgctcctaa caaatgaaga agcaggctgc agcctgcatt gtggaagatc 3109tccagcctcc ttgtagggga taaggggatg tgtagcatct gtgtggattt tcacggacaa 3169gttccagtag gtgggacagt gatgccgtca aggcttagtt atgatcatgt gtggtgataa 3229agaccatcca ccatcaccct tttccccttt ggttttgaag gccttgccct aagctacctg 3289agggtttagg aggtctgaac acacacagtg gagaggttaa tctaggttgg gaaactgagt 3349aaaagtccag agcaggaatg agcctgctgt ggcgtgggtt tggaaaggct cacaggaaag 3409aacctgcagg atcaggggtg ggaggggagg cccctgaggt gctctccagg gaagaggggc 3469tggggtttaa atagcatgct tggaggaaga ttttccttca atttttccta agtccttgaa 3529ttcaccagta gatttttgta aacaaaatgt aagtcgatgt tttctctcaa ttatcctagg 3589agtgaccttt atatgtgtgg aagattaatg gtatatgctc cttatgtcac tgtttttgag 3649taaaatccat ttcctttctc tgtttcagcc tatgacaaaa ttgatgttta caggcctgct 3709ttttgcttat aattgacaac atgtgcaaaa ataccaaatt tgtgtcctgt gcagtatgaa 3769gaattcagtg aatattcatt aatgtattag cttgttttgc tctctgttca tatatggctc 3829tattcttaga aatataattt gaatgtgatc tttcaatagt ctgaatattt tacaaattat 3889agctatgtct tgtgaaaata acctcaaaaa gaaaaatacg actctgttgt cttacttgat 3949atttcttgcc ctagtaatgt acttgacatt tatgttccta agcagtgtaa gtaccagtag 4009aatttctctg tcaaactcaa tgatcattta gtacttttgt cttctcccat gtgcttgaag 4069gaaaaataaa gtgtcactac cgtatttctt gttttcatca aaaaataaaa ataatttaaa 4129aaacaaaaaa aaaaaaaaa 4148 55 414 PRT Homo sapiens 55 Met Ser Gly Leu AspGly Gly Asn Lys Leu Pro Leu Ala Gln Thr Gly 1 5 10 15 Gly Leu Ala AlaPro Asp His Ala Ser Gly Asp Pro Asp Leu Asp Gln 20 25 30 Cys Gln Gly LeuArg Glu Glu Thr Glu Ala Thr Gln Val Met Ala Asn 35 40 45 Thr Gly Gly GlySer Leu Glu Thr Val Ala Glu Gly Gly Ala Ser Gln 50 55 60 Asp Pro Val AspCys Gly Pro Ala Leu Arg Val Pro Val Ala Gly Ser 65 70 75 80 Arg Gly GlyAla Ala Thr Lys Ala Gly Gln Glu Asp Ala Pro Pro Ser 85 90 95 Thr Lys GlyLeu Glu Ala Ala Ser Ala Ala Glu Ala Ala Asp Ser Ser 100 105 110 Gln LysAsn Gly Cys Gln Leu Gly Glu Pro Arg Gly Pro Ala Gly Gln 115 120 125 LysAla Leu Glu Ala Cys Gly Ala Gly Gly Leu Gly Ser Gln Met Ile 130 135 140Pro Gly Lys Lys Ala Lys Glu Val Thr Thr Lys Lys Arg Ala Ile Ser 145 150155 160 Ala Ala Val Glu Lys Glu Gly Glu Ala Gly Ala Ala Met Glu Glu Lys165 170 175 Lys Val Val Gln Lys Glu Lys Lys Val Ala Gly Gly Val Lys GluGlu 180 185 190 Thr Arg Pro Arg Ala Pro Lys Ile Asn Asn Cys Met Asp SerLeu Glu 195 200 205 Ala Ile Asp Gln Glu Leu Ser Asn Val Asn Ala Gln AlaAsp Arg Ala 210 215 220 Phe Leu Gln Leu Glu Arg Lys Phe Gly Arg Met ArgArg Leu His Met 225 230 235 240 Gln Arg Arg Ser Phe Ile Ile Gln Asn IlePro Gly Phe Trp Val Thr 245 250 255 Ala Phe Arg Asn His Pro Gln Leu SerPro Met Ile Ser Gly Gln Asp 260 265 270 Glu Asp Met Leu Arg Tyr Met IleAsn Leu Glu Val Glu Glu Leu Lys 275 280 285 His Pro Arg Ala Gly Cys LysPhe Lys Phe Ile Phe Gln Gly Asn Pro 290 295 300 Tyr Phe Arg Asn Glu GlyLeu Val Lys Glu Tyr Glu Arg Arg Ser Ser 305 310 315 320 Gly Arg Val ValSer Leu Ser Thr Pro Ile Arg Trp His Arg Gly Gln 325 330 335 Asp Pro GlnAla His Ile His Arg Asn Arg Glu Gly Asn Thr Ile Pro 340 345 350 Ser PhePhe Asn Trp Phe Ser Asp His Ser Leu Leu Glu Phe Asp Arg 355 360 365 IleAla Glu Ile Ile Lys Gly Glu Leu Trp Pro Asn Pro Leu Gln Tyr 370 375 380Tyr Leu Met Gly Glu Gly Pro Arg Arg Gly Ile Arg Gly Pro Pro Arg 385 390395 400 Gln Pro Val Glu Ser Ala Arg Ser Phe Arg Phe Gln Ser Gly 405 41056 2643 DNA Rattus sp. CDS (1)..(801) 56 ctg aaa ggg gcg agg ccc agg gtggtg aac tcc acc tgc agt gac ttc 48 Leu Lys Gly Ala Arg Pro Arg Val ValAsn Ser Thr Cys Ser Asp Phe 1 5 10 15 aac cat ggc tca gct ctg cac atcgct gcc tcg aat ctg tgc ctg ggc 96 Asn His Gly Ser Ala Leu His Ile AlaAla Ser Asn Leu Cys Leu Gly 20 25 30 gcc gcc aaa tgt tta ctg gag cat ggtgcc aac cca gcg ctg agg aat 144 Ala Ala Lys Cys Leu Leu Glu His Gly AlaAsn Pro Ala Leu Arg Asn 35 40 45 cga aaa gga cag gta cca gcg gaa gtg gtccca gac ccc atg gac atg 192 Arg Lys Gly Gln Val Pro Ala Glu Val Val ProAsp Pro Met Asp Met 50 55 60 tcc ctt gac aag gca gag gca gcc ctg gtg gccaag gaa ttg cgg acg 240 Ser Leu Asp Lys Ala Glu Ala Ala Leu Val Ala LysGlu Leu Arg Thr 65 70 75 80 ctg cta gaa gag gct gtg cca ctg tcc tgc accctt cct aaa gtc aca 288 Leu Leu Glu Glu Ala Val Pro Leu Ser Cys Thr LeuPro Lys Val Thr 85 90 95 cta ccc aac tat gac aac gtc cca ggc aat ctc atgctc agc gcg ctg 336 Leu Pro Asn Tyr Asp Asn Val Pro Gly Asn Leu Met LeuSer Ala Leu 100 105 110 ggc ctg cgt cta gga gac cga gtg ctc ctc gat ggccag aag acg ggc 384 Gly Leu Arg Leu Gly Asp Arg Val Leu Leu Asp Gly GlnLys Thr Gly 115 120 125 acg ctg agg ttc tgc ggg acc acc gag ttc gcc agtggc cag tgg gtg 432 Thr Leu Arg Phe Cys Gly Thr Thr Glu Phe Ala Ser GlyGln Trp Val 130 135 140 ggc gtg gag cta gat gaa ccg gaa ggc aag aac gacggc agc gtt ggg 480 Gly Val Glu Leu Asp Glu Pro Glu Gly Lys Asn Asp GlySer Val Gly 145 150 155 160 ggt gtc cgg tac ttc atc tgc cct ccc aag cagggt ctc ttt gca tct 528 Gly Val Arg Tyr Phe Ile Cys Pro Pro Lys Gln GlyLeu Phe Ala Ser 165 170 175 gtg tcc aag gtc tcc aag gca gtg gat gca cccccc tca tct gtt acc 576 Val Ser Lys Val Ser Lys Ala Val Asp Ala Pro ProSer Ser Val Thr 180 185 190 tcc acg ccc cgc act ccc cgg atg gac ttc tcccgt gta acg ggc aaa 624 Ser Thr Pro Arg Thr Pro Arg Met Asp Phe Ser ArgVal Thr Gly Lys 195 200 205 ggc cgg agg gaa cac aaa ggg aag aag aag tcccca tct tcc cca tct 672 Gly Arg Arg Glu His Lys Gly Lys Lys Lys Ser ProSer Ser Pro Ser 210 215 220 ctg ggc agc ctg cag cag cgt gaa ggg gcc aaagct gaa gtt gga gac 720 Leu Gly Ser Leu Gln Gln Arg Glu Gly Ala Lys AlaGlu Val Gly Asp 225 230 235 240 caa gtc ctt gtg gca ggc cag aac agg gattgt gcg ttt cta tgg gaa 768 Gln Val Leu Val Ala Gly Gln Asn Arg Asp CysAla Phe Leu Trp Glu 245 250 255 gac aga ctt tgc tcc agg tta ctg gta tggcat tgaactggac cagcccacgg 821 Asp Arg Leu Cys Ser Arg Leu Leu Val TrpHis 260 265 gcaagcatga cggctctgtg ttcggtgtcc ggtactttac ctgtgccccgaggcacgggg 881 tctttgcacc agcatctcgt atccagagga ttggtggatc cactgatccccctggagaca 941 gtgttggagc aaaaaaagtg catcaagtga caatgacaca gcccaaacgcaccttcacaa 1001 cagtccggac cccaaaggac attgcatcag agaactctat ctccaggttactcttctgct 1061 gctggtttcc ttggatgctg agggcggaga tgcagtctta gagacctggatacctgacac 1121 agagacagag tcccctctag catctcctga cacaaggaga ccccagtcaccctaagatag 1181 agattcccag tgacacctcc agaatagaaa ccccgttagc cagccctcgattactgaggt 1241 cccattatta acagatctcc catgacgact cccccaaata cagacctcatgttaccccaa 1301 aagagattcc ctgagtagca ccttcaggct agtccctgtc ccctacccctcagagcagat 1361 ttcccccaat aaacattttc cacatcaccc aagggatgct gaccctctccacgacaggac 1421 gttcttgagt taccagtgga ttagagtccc atgaatgaag accccccccaccccggttct 1481 ccttaagcat aggtcatacc tccagaatag ccagccacat cactatccccatgtaacatc 1541 agtctcctca aaatggcgtg aggtcactag aaagacctta tactctcctctccttctcag 1601 agatgccctc cattcactta agtccctgtt ctcacccctg aacaagacacctaattaacc 1661 ggcccactca cctcaattac aaacaccaaa atcgtcctgg aagcatgaattacaggacag 1721 caagtcttcc tgccctctgc acccttgaga aacccccagt gccttgtatgaagcccaccc 1781 cacatggccc acagtccctg tgctggccaa ggctcccaga aaattctctattttttaaag 1841 taataacttc cccccctttg gggggatccc caaatttgga gaccccattctagaacactg 1901 gggagttcaa attccagaga gaatatatat tatatataat ccccaattccccatgcttcc 1961 aagccctaca atctctagaa gaccccaaat ttctaattcc caggacttcccctacccaag 2021 tcacagaatc ttcaaatccc cagggaatcc caaacttaag ataccaatcccaaaccctca 2081 ggaaatcccc caacacaagg tccttaggac cgggaggaag gaacctgttgccaggagaac 2141 atcccaggct ctcagggcat ctcaaacctg actcccaggc accaggagaccccaaacaga 2201 aagtcccatc tttggaacaa ggataggact ctaataccct tagtccatggatctttaatt 2261 tcccaacctc caaactccat gggccccacc ctcaagggaa cccccaagatccaaatctct 2321 gataactaat atgtgcaggg ccccagggct ctaacaggac cccaaatcatggagtcccta 2381 cttcaatcta ccttctggtc acaggtccaa gacactaaat ctgagtcattggccccaaag 2441 gacttcacag cacctgggcc agactaacag cctgagggag aacctgagggccccgtgggt 2501 ccagagcaga cctggggccc tgaccaccaa ggacagctca cgactgccccttcactgcat 2561 gtccctaaac tcagcatgac tcctgtcctc ttcaataaag acgtttctatggcaaaaaaa 2621 aaaaaaaaaa aaaaaaaaaa aa 2643 57 267 PRT Rattus sp. 57Leu Lys Gly Ala Arg Pro Arg Val Val Asn Ser Thr Cys Ser Asp Phe 1 5 1015 Asn His Gly Ser Ala Leu His Ile Ala Ala Ser Asn Leu Cys Leu Gly 20 2530 Ala Ala Lys Cys Leu Leu Glu His Gly Ala Asn Pro Ala Leu Arg Asn 35 4045 Arg Lys Gly Gln Val Pro Ala Glu Val Val Pro Asp Pro Met Asp Met 50 5560 Ser Leu Asp Lys Ala Glu Ala Ala Leu Val Ala Lys Glu Leu Arg Thr 65 7075 80 Leu Leu Glu Glu Ala Val Pro Leu Ser Cys Thr Leu Pro Lys Val Thr 8590 95 Leu Pro Asn Tyr Asp Asn Val Pro Gly Asn Leu Met Leu Ser Ala Leu100 105 110 Gly Leu Arg Leu Gly Asp Arg Val Leu Leu Asp Gly Gln Lys ThrGly 115 120 125 Thr Leu Arg Phe Cys Gly Thr Thr Glu Phe Ala Ser Gly GlnTrp Val 130 135 140 Gly Val Glu Leu Asp Glu Pro Glu Gly Lys Asn Asp GlySer Val Gly 145 150 155 160 Gly Val Arg Tyr Phe Ile Cys Pro Pro Lys GlnGly Leu Phe Ala Ser 165 170 175 Val Ser Lys Val Ser Lys Ala Val Asp AlaPro Pro Ser Ser Val Thr 180 185 190 Ser Thr Pro Arg Thr Pro Arg Met AspPhe Ser Arg Val Thr Gly Lys 195 200 205 Gly Arg Arg Glu His Lys Gly LysLys Lys Ser Pro Ser Ser Pro Ser 210 215 220 Leu Gly Ser Leu Gln Gln ArgGlu Gly Ala Lys Ala Glu Val Gly Asp 225 230 235 240 Gln Val Leu Val AlaGly Gln Asn Arg Asp Cys Ala Phe Leu Trp Glu 245 250 255 Asp Arg Leu CysSer Arg Leu Leu Val Trp His 260 265 58 2929 DNA Rattus sp. CDS(1)..(810) 58 gct gac tct acc tct aga tgg gct gag gcc ctc aga gaa atctct ggt 48 Ala Asp Ser Thr Ser Arg Trp Ala Glu Ala Leu Arg Glu Ile SerGly 1 5 10 15 cgc tta gct gaa atg cct gca gat agt gga tac cct gca tacctt ggt 96 Arg Leu Ala Glu Met Pro Ala Asp Ser Gly Tyr Pro Ala Tyr LeuGly 20 25 30 gcc cga ctg gct tct ttc tat gag cga gca ggc aga gtg aaa tgtctt 144 Ala Arg Leu Ala Ser Phe Tyr Glu Arg Ala Gly Arg Val Lys Cys Leu35 40 45 gga aac cct gag aga gaa ggg agt gtc agc att gta gga gca gtt tct192 Gly Asn Pro Glu Arg Glu Gly Ser Val Ser Ile Val Gly Ala Val Ser 5055 60 cca cct ggt ggt gat ttt tct gat cca gtc aca tct gct act ctg ggt240 Pro Pro Gly Gly Asp Phe Ser Asp Pro Val Thr Ser Ala Thr Leu Gly 6570 75 80 att gtt cag gtg ttc tgg ggc ttg gat aag aag cta gct cag cgc aag288 Ile Val Gln Val Phe Trp Gly Leu Asp Lys Lys Leu Ala Gln Arg Lys 8590 95 cac ttc ccg tcc gtc aac tgg ctc att agc tac agc aag tac atg cgc336 His Phe Pro Ser Val Asn Trp Leu Ile Ser Tyr Ser Lys Tyr Met Arg 100105 110 gcc ctg gac gag tac tat gac aaa cac ttc aca gag ttc gtg cct ctg384 Ala Leu Asp Glu Tyr Tyr Asp Lys His Phe Thr Glu Phe Val Pro Leu 115120 125 agg acc aaa gct aag gag att ctg cag gaa gag gag gat ctg gcg gaa432 Arg Thr Lys Ala Lys Glu Ile Leu Gln Glu Glu Glu Asp Leu Ala Glu 130135 140 atc gtg cag ctc gtg gga aag gcg tct tta gca gag aca gat aaa atc480 Ile Val Gln Leu Val Gly Lys Ala Ser Leu Ala Glu Thr Asp Lys Ile 145150 155 160 acc ctg gag gta gca aaa ctt atc aaa gat gac ttc cta caa caaaat 528 Thr Leu Glu Val Ala Lys Leu Ile Lys Asp Asp Phe Leu Gln Gln Asn165 170 175 ggg tac act cct tat gac agg ttc tgt cca ttc tat aag acg gtgggg 576 Gly Tyr Thr Pro Tyr Asp Arg Phe Cys Pro Phe Tyr Lys Thr Val Gly180 185 190 atg ctg tcc aac atg att tca ttc tat gat atg gcc cgc cgg gctgtg 624 Met Leu Ser Asn Met Ile Ser Phe Tyr Asp Met Ala Arg Arg Ala Val195 200 205 gag acc acc gcc cag agt gac aat aag atc aca tgg tcc att atccgt 672 Glu Thr Thr Ala Gln Ser Asp Asn Lys Ile Thr Trp Ser Ile Ile Arg210 215 220 gag cac atg ggg gag att ctc tat aaa ctt tcc tcc atg aaa ttcaag 720 Glu His Met Gly Glu Ile Leu Tyr Lys Leu Ser Ser Met Lys Phe Lys225 230 235 240 gat cca gtg aag gat ggc gag gca aag atc aag gcc gac tacgca cag 768 Asp Pro Val Lys Asp Gly Glu Ala Lys Ile Lys Ala Asp Tyr AlaGln 245 250 255 ctt ctt gaa gat atg cag aac gca ttc cgt agc ctg gaa gat810 Leu Leu Glu Asp Met Gln Asn Ala Phe Arg Ser Leu Glu Asp 260 265 270tagaactgtg acttctctcc tcctcttccg cagctcatat gtgtatattt tcctgaattt 870ctcatctcca accctttgct tccatattgt gcagctttga gactagtgcc tcgtgcgttc 930tcgttcattt tgctgtttct ttggtaggtc ttataaaaca cacattcctg tgctccgctg 990tctgaaggag ctcctgacct ttgtctgaag tggtgaatgt agtgcatatg atacacagtg 1050taacatacac attgtaacat atacgttctg taaacttgta tgtaaggtga ctaccccttc 1110cctcctctcc agtaaactgt aaacaggact actgcatgtg ctctattggg gatggaaggc 1170cagatctcca taccgtggac aggtacataa ggaaactaga ccacttgcaa cttagtgttt 1230gttgagtaac cattttgcag gaagtatttc catttaaaaa acaaaagatt aatgttccaa 1290ttatttgtag cttccccagt atcaatcagg actgtttgtg gcgcacttgg gaactatttt 1350gttttcctaa cagacgtttg caaggctgaa cgtaatagat aaatcagttc cctctgaaag 1410tgtgaaagta aaaagagagc taggtggtca gacttaaatt gacatcgtct tgtttaagca 1470tattttattt cactgagaga tttaatatca aggactttta tatactcaat tactaggaaa 1530tcttttttta agtacaattt aaaaatcatt gaaaatgtga tccacatcat agccattttc 1590cttatattta gtcagatgag ctcagagtgg ggagggtgtg ggttagaata ccacaaggac 1650acgcagcagt gcctgcaggc agtgtggccg ggggccagag cggcattgtt ttcacgaggt 1710acgtgtgtgg cgtgtgtgtt tgcttgttga cactctgaaa acagcaagct taccagttcc 1770aggaaatatt ttgttttctt tcactggctc agaaagctcc tcaaagtacc tggtccctga 1830agcttcctat ctgttaatag agacgagaga ggttcttaaa tttaactggt gacaaaacaa 1890aaagaaaaaa aagatcgatt tttgtcttgc tgttttggtg tgtttaaata ataattccat 1950atttgcataa cgaggctcgc ttctgagagc ttggagatcg tgctccctct tcactctccg 2010gggtgataat gctggcgcca tgctacctct tcaggagggg aaggggattg aacatggcta 2070acactctcaa gtacacaagc gtaacgacaa agtatttatt ttaagccttg gtatgttgtt 2130taaattatta ggtggtgcat ttcttatggt cttttgggta gacatagtat acacttcaga 2190tgtaatgtgt aaatccttgc tagtgcatgt ctacacgata gactgctatt caagaaggat 2250attcttccac ataacaattt aaaaactatt aaatcagata tggattatgc aatgacttgt 2310tgagaggtgg attaacggtg ctgcttaatc agtttgcttc caatatggct tcgtatccag 2370aagccctgac tagtggagat gagaaagatt tcaaaacctg tctgcctaca cctaccagca 2430acctaggctt gtgatcagaa tgaatgatcc caagaaacta cttgaccaag tgtgttttgt 2490tgtcctggat ttgagatgtg cgttcttcct ccctctgaga ctgttgatgt atgagtgtga 2550agaagttaca gaaacaacgc tcagattttc acggtaactt tccctctgcc cacactgtag 2610agtttcagat tgttcactga tagtgcttct ttcgtaagga tgtgttaaaa tatagcagtc 2670tttttaaaag attatgcagt tctctattta ttgtgctgtg cctggtccta agtgcagccg 2730gttaaacaag tttcatatgt atttttccag tgttaaatct catacctatg ccctttggaa 2790agctccatcc tgaacaatga atagaagagg ctatataaat tgcctcctta tccttaagat 2850ttcactatct ttatgttaag agtaatgtat aattattaaa atctatgaaa aataaaaagt 2910ggatttaaat taagagatc 2929 59 270 PRT Rattus sp. 59 Ala Asp Ser Thr SerArg Trp Ala Glu Ala Leu Arg Glu Ile Ser Gly 1 5 10 15 Arg Leu Ala GluMet Pro Ala Asp Ser Gly Tyr Pro Ala Tyr Leu Gly 20 25 30 Ala Arg Leu AlaSer Phe Tyr Glu Arg Ala Gly Arg Val Lys Cys Leu 35 40 45 Gly Asn Pro GluArg Glu Gly Ser Val Ser Ile Val Gly Ala Val Ser 50 55 60 Pro Pro Gly GlyAsp Phe Ser Asp Pro Val Thr Ser Ala Thr Leu Gly 65 70 75 80 Ile Val GlnVal Phe Trp Gly Leu Asp Lys Lys Leu Ala Gln Arg Lys 85 90 95 His Phe ProSer Val Asn Trp Leu Ile Ser Tyr Ser Lys Tyr Met Arg 100 105 110 Ala LeuAsp Glu Tyr Tyr Asp Lys His Phe Thr Glu Phe Val Pro Leu 115 120 125 ArgThr Lys Ala Lys Glu Ile Leu Gln Glu Glu Glu Asp Leu Ala Glu 130 135 140Ile Val Gln Leu Val Gly Lys Ala Ser Leu Ala Glu Thr Asp Lys Ile 145 150155 160 Thr Leu Glu Val Ala Lys Leu Ile Lys Asp Asp Phe Leu Gln Gln Asn165 170 175 Gly Tyr Thr Pro Tyr Asp Arg Phe Cys Pro Phe Tyr Lys Thr ValGly 180 185 190 Met Leu Ser Asn Met Ile Ser Phe Tyr Asp Met Ala Arg ArgAla Val 195 200 205 Glu Thr Thr Ala Gln Ser Asp Asn Lys Ile Thr Trp SerIle Ile Arg 210 215 220 Glu His Met Gly Glu Ile Leu Tyr Lys Leu Ser SerMet Lys Phe Lys 225 230 235 240 Asp Pro Val Lys Asp Gly Glu Ala Lys IleLys Ala Asp Tyr Ala Gln 245 250 255 Leu Leu Glu Asp Met Gln Asn Ala PheArg Ser Leu Glu Asp 260 265 270 60 1489 DNA Rattus sp. CDS (1)..(1053)60 gca cgg ctc ccg gcc ccg gag cat gcg cga cag cag ccc ctc ctc tcc 48Ala Arg Leu Pro Ala Pro Glu His Ala Arg Gln Gln Pro Leu Leu Ser 1 5 1015 ggc cct gag ccc gga tcg tcc gcc cgg gtt cca gtt ccc ggc gtg gcc 96Gly Pro Glu Pro Gly Ser Ser Ala Arg Val Pro Val Pro Gly Val Ala 20 25 30agt agg cgg cag ccg cga ggc ggc aag cca ccc agc ggg gac ggc ctg 144 SerArg Arg Gln Pro Arg Gly Gly Lys Pro Pro Ser Gly Asp Gly Leu 35 40 45 gagtcg ggc ccc tct cca cgc ccc ctt ctc cac gcg cgc ggg gag gca 192 Glu SerGly Pro Ser Pro Arg Pro Leu Leu His Ala Arg Gly Glu Ala 50 55 60 ggg ctccac cgc cag tct gga agg gtt cca cat aca gga acg gcc tac 240 Gly Leu HisArg Gln Ser Gly Arg Val Pro His Thr Gly Thr Ala Tyr 65 70 75 80 ttc gcagat gag ccc acc gag gct cag gct ccg ggc gga ttc tgc gtg 288 Phe Ala AspGlu Pro Thr Glu Ala Gln Ala Pro Gly Gly Phe Cys Val 85 90 95 tca ccc tcgctc ctt ggg gtc cgc tgg ccg gcc tgt gcc acc cgg acg 336 Ser Pro Ser LeuLeu Gly Val Arg Trp Pro Ala Cys Ala Thr Arg Thr 100 105 110 ccc ggc tcactg cct ctg tct ccc cca tca gcg cag ccc cgg acg cta 384 Pro Gly Ser LeuPro Leu Ser Pro Pro Ser Ala Gln Pro Arg Thr Leu 115 120 125 tgg ccc acccct cca gct ggc ccc tcg agt agg atg gta gca cgt aac 432 Trp Pro Thr ProPro Ala Gly Pro Ser Ser Arg Met Val Ala Arg Asn 130 135 140 cag gtg gcagcc gac aat gcg atc tcc ccg gca tca gag ccc cga cgg 480 Gln Val Ala AlaAsp Asn Ala Ile Ser Pro Ala Ser Glu Pro Arg Arg 145 150 155 160 cgg ccagag cca tcc tcg tcc tcg tct tcg tcc tcg ccg gcg gcc ccg 528 Arg Pro GluPro Ser Ser Ser Ser Ser Ser Ser Ser Pro Ala Ala Pro 165 170 175 gcg cgtccc cgg ccc tgc ccg gtg gtc ccg gcc ccg gct ccg ggc gac 576 Ala Arg ProArg Pro Cys Pro Val Val Pro Ala Pro Ala Pro Gly Asp 180 185 190 act cacttc cgc acc ttc cgc tcc cac tct gat tac cgg cgc atc acg 624 Thr His PheArg Thr Phe Arg Ser His Ser Asp Tyr Arg Arg Ile Thr 195 200 205 cgg accagc gct ctc ctg gac gcc tgc ggc ttc tac tgg gga ccc ctg 672 Arg Thr SerAla Leu Leu Asp Ala Cys Gly Phe Tyr Trp Gly Pro Leu 210 215 220 agc gtgcat ggg gcg cac gaa cgg ctg cgt gcc gag ccc gtg ggc acc 720 Ser Val HisGly Ala His Glu Arg Leu Arg Ala Glu Pro Val Gly Thr 225 230 235 240 ttcttg gtg cgc gac agt cgc cag cgg aac tgc ttc ttc gcg ctc agc 768 Phe LeuVal Arg Asp Ser Arg Gln Arg Asn Cys Phe Phe Ala Leu Ser 245 250 255 gtgaag atg gct tcg ggc ccc acg agc att cgt gtg cac ttc cag gcc 816 Val LysMet Ala Ser Gly Pro Thr Ser Ile Arg Val His Phe Gln Ala 260 265 270 ggccgc ttc cac ctg gac ggc agc cgc gag acc ttc gac tgc ctc ttc 864 Gly ArgPhe His Leu Asp Gly Ser Arg Glu Thr Phe Asp Cys Leu Phe 275 280 285 gagctg ctg gag cac tac gtg gcg gcg ccg cgc cgc atg ttg ggg gcc 912 Glu LeuLeu Glu His Tyr Val Ala Ala Pro Arg Arg Met Leu Gly Ala 290 295 300 ccactg cgc cag cgc cgc gtg cgg ccg ctg cag gag ctg tgt cgc cag 960 Pro LeuArg Gln Arg Arg Val Arg Pro Leu Gln Glu Leu Cys Arg Gln 305 310 315 320cgc atc gtg gcc gcc gtg ggt cgc gag aac ctg gca cgc atc cct ctt 1008 ArgIle Val Ala Ala Val Gly Arg Glu Asn Leu Ala Arg Ile Pro Leu 325 330 335aac ccg gta ctc cgt gac tac ctg agt tcc ttc ccc ttc cag atc 1053 Asn ProVal Leu Arg Asp Tyr Leu Ser Ser Phe Pro Phe Gln Ile 340 345 350tgaccggctg ccgccgtgcc cgcagcatta agtgggagcg ccttattatt tcttattatt 1113aattattatt atttttctgg aaccacgtgg gagccctccc cgcctaggtc ggagggagtg 1173ggtgtggagg gtgagatgcc tcccacttct ggctggagac cttatcccgc ctctcggggg 1233gcctcccctc ctggtgctcc ctcccggtcc ccctggttgt agcagcttgt gtctggggcc 1293aggacctgaa ctccacgcct acctctccat gtttacatgt tcccagtatc tttgcacaaa 1353ccaggggtgg gggagggtct ctggcttcat ttttctgctg tgcagaatat tctattttat 1413atttttacat ccagtttaga taataaactt tattatgaaa gttttttttt taaagaaaaa 1473aaaaaaaaaa aaaaaa 1489 61 351 PRT Rattus sp. 61 Ala Arg Leu Pro Ala ProGlu His Ala Arg Gln Gln Pro Leu Leu Ser 1 5 10 15 Gly Pro Glu Pro GlySer Ser Ala Arg Val Pro Val Pro Gly Val Ala 20 25 30 Ser Arg Arg Gln ProArg Gly Gly Lys Pro Pro Ser Gly Asp Gly Leu 35 40 45 Glu Ser Gly Pro SerPro Arg Pro Leu Leu His Ala Arg Gly Glu Ala 50 55 60 Gly Leu His Arg GlnSer Gly Arg Val Pro His Thr Gly Thr Ala Tyr 65 70 75 80 Phe Ala Asp GluPro Thr Glu Ala Gln Ala Pro Gly Gly Phe Cys Val 85 90 95 Ser Pro Ser LeuLeu Gly Val Arg Trp Pro Ala Cys Ala Thr Arg Thr 100 105 110 Pro Gly SerLeu Pro Leu Ser Pro Pro Ser Ala Gln Pro Arg Thr Leu 115 120 125 Trp ProThr Pro Pro Ala Gly Pro Ser Ser Arg Met Val Ala Arg Asn 130 135 140 GlnVal Ala Ala Asp Asn Ala Ile Ser Pro Ala Ser Glu Pro Arg Arg 145 150 155160 Arg Pro Glu Pro Ser Ser Ser Ser Ser Ser Ser Ser Pro Ala Ala Pro 165170 175 Ala Arg Pro Arg Pro Cys Pro Val Val Pro Ala Pro Ala Pro Gly Asp180 185 190 Thr His Phe Arg Thr Phe Arg Ser His Ser Asp Tyr Arg Arg IleThr 195 200 205 Arg Thr Ser Ala Leu Leu Asp Ala Cys Gly Phe Tyr Trp GlyPro Leu 210 215 220 Ser Val His Gly Ala His Glu Arg Leu Arg Ala Glu ProVal Gly Thr 225 230 235 240 Phe Leu Val Arg Asp Ser Arg Gln Arg Asn CysPhe Phe Ala Leu Ser 245 250 255 Val Lys Met Ala Ser Gly Pro Thr Ser IleArg Val His Phe Gln Ala 260 265 270 Gly Arg Phe His Leu Asp Gly Ser ArgGlu Thr Phe Asp Cys Leu Phe 275 280 285 Glu Leu Leu Glu His Tyr Val AlaAla Pro Arg Arg Met Leu Gly Ala 290 295 300 Pro Leu Arg Gln Arg Arg ValArg Pro Leu Gln Glu Leu Cys Arg Gln 305 310 315 320 Arg Ile Val Ala AlaVal Gly Arg Glu Asn Leu Ala Arg Ile Pro Leu 325 330 335 Asn Pro Val LeuArg Asp Tyr Leu Ser Ser Phe Pro Phe Gln Ile 340 345 350 62 1194 DNARattus sp. CDS (130)..(765) 62 ggcacggctc ccggccccgg agcatgcgcgacagcagccc cggaaccccc agccgcggcg 60 ccccgcgtcc cgccgccagc gcagccccggacgctatggc ccacccctcc agctggcccc 120 tcgagtagg atg gta gca cgt aac caggtg gca gcc gac aat gcg atc tcc 171 Met Val Ala Arg Asn Gln Val Ala AlaAsp Asn Ala Ile Ser 1 5 10 ccg gca tca gag ccc cga cgg cgg cca gag ccatcc tcg tcc tcg tct 219 Pro Ala Ser Glu Pro Arg Arg Arg Pro Glu Pro SerSer Ser Ser Ser 15 20 25 30 tcg tcc tcg ccg gcg gcc ccg gcg cgt ccc cggccc tgc ccg gtg gtc 267 Ser Ser Ser Pro Ala Ala Pro Ala Arg Pro Arg ProCys Pro Val Val 35 40 45 ccg gcc ccg gct ccg ggc gac act cac ttc cgc accttc cgc tcc cac 315 Pro Ala Pro Ala Pro Gly Asp Thr His Phe Arg Thr PheArg Ser His 50 55 60 tct gat tac cgg cgc atc acg cgg acc agc gct ctc ctggac gcc tgc 363 Ser Asp Tyr Arg Arg Ile Thr Arg Thr Ser Ala Leu Leu AspAla Cys 65 70 75 ggc ttc tac tgg gga ccc ctg agc gtg cat ggg gcg cac gaacgg ctg 411 Gly Phe Tyr Trp Gly Pro Leu Ser Val His Gly Ala His Glu ArgLeu 80 85 90 cgt gcc gag ccc gtg ggc acc ttc ttg gtg cgc gac agt cgc cagcgg 459 Arg Ala Glu Pro Val Gly Thr Phe Leu Val Arg Asp Ser Arg Gln Arg95 100 105 110 aac tgc ttc ttc gcg ctc agc gtg aag atg gct tcg ggc cccacg agc 507 Asn Cys Phe Phe Ala Leu Ser Val Lys Met Ala Ser Gly Pro ThrSer 115 120 125 att cgt gtg cac ttc cag gcc ggc cgc ttc cac ctg gac ggcagc cgc 555 Ile Arg Val His Phe Gln Ala Gly Arg Phe His Leu Asp Gly SerArg 130 135 140 gag acc ttc gac tgc ctc ttc gag ctg ctg gag cac tac gtggcg gcg 603 Glu Thr Phe Asp Cys Leu Phe Glu Leu Leu Glu His Tyr Val AlaAla 145 150 155 ccg cgc cgc atg ttg ggg gcc cca ctg cgc cag cgc cgc gtgcgg ccg 651 Pro Arg Arg Met Leu Gly Ala Pro Leu Arg Gln Arg Arg Val ArgPro 160 165 170 ctg cag gag ctg tgt cgc cag cgc atc gtg gcc gcc gtg ggtcgc gag 699 Leu Gln Glu Leu Cys Arg Gln Arg Ile Val Ala Ala Val Gly ArgGlu 175 180 185 190 aac ctg gca cgc atc cct ctt aac ccg gta ctc cgt gactac ctg agt 747 Asn Leu Ala Arg Ile Pro Leu Asn Pro Val Leu Arg Asp TyrLeu Ser 195 200 205 tcc ttc ccc ttc cag atc tgaccggctg ccgccgtgcccgcagcatta 795 Ser Phe Pro Phe Gln Ile 210 agtgggagcg ccttattatttcttattatt aattattatt atttttctgg aaccacgtgg 855 gagccctccc cgcctaggtcggagggagtg ggtgtggagg gtgagatgcc tcccacttct 915 ggctggagac cttatcccgcctctcggggg gcctcccctc ctggtgctcc ctcccggtcc 975 ccctggttgt agcagcttgtgtctggggcc aggacctgaa ctccacgcct acctctccat 1035 gtttacatgt tcccagtatctttgcacaaa ccaggggtgg gggagggtct ctggcttcat 1095 ttttctgctg tgcagaatattctattttat atttttacat ccagtttaga taataaactt 1155 tattatgaaa gttttttttttaaaaaaaaa aaaaaaaaa 1194 63 212 PRT Rattus sp. 63 Met Val Ala Arg AsnGln Val Ala Ala Asp Asn Ala Ile Ser Pro Ala 1 5 10 15 Ser Glu Pro ArgArg Arg Pro Glu Pro Ser Ser Ser Ser Ser Ser Ser 20 25 30 Ser Pro Ala AlaPro Ala Arg Pro Arg Pro Cys Pro Val Val Pro Ala 35 40 45 Pro Ala Pro GlyAsp Thr His Phe Arg Thr Phe Arg Ser His Ser Asp 50 55 60 Tyr Arg Arg IleThr Arg Thr Ser Ala Leu Leu Asp Ala Cys Gly Phe 65 70 75 80 Tyr Trp GlyPro Leu Ser Val His Gly Ala His Glu Arg Leu Arg Ala 85 90 95 Glu Pro ValGly Thr Phe Leu Val Arg Asp Ser Arg Gln Arg Asn Cys 100 105 110 Phe PheAla Leu Ser Val Lys Met Ala Ser Gly Pro Thr Ser Ile Arg 115 120 125 ValHis Phe Gln Ala Gly Arg Phe His Leu Asp Gly Ser Arg Glu Thr 130 135 140Phe Asp Cys Leu Phe Glu Leu Leu Glu His Tyr Val Ala Ala Pro Arg 145 150155 160 Arg Met Leu Gly Ala Pro Leu Arg Gln Arg Arg Val Arg Pro Leu Gln165 170 175 Glu Leu Cys Arg Gln Arg Ile Val Ala Ala Val Gly Arg Glu AsnLeu 180 185 190 Ala Arg Ile Pro Leu Asn Pro Val Leu Arg Asp Tyr Leu SerSer Phe 195 200 205 Pro Phe Gln Ile 210 64 600 DNA Rattus sp. CDS(52)..(336) 64 cttccaaaga ctgcagcgcc tcagggccca ggtttcaaca gattcttcaa aatg cca 57 Met Pro 1 tcc caa atg gag cat gcc atg gaa acc atg atg ctt acattt cac agg 105 Ser Gln Met Glu His Ala Met Glu Thr Met Met Leu Thr PheHis Arg 5 10 15 ttt gca ggg gaa aaa aac tac ttg aca aag gag gac ctg agagtg ctc 153 Phe Ala Gly Glu Lys Asn Tyr Leu Thr Lys Glu Asp Leu Arg ValLeu 20 25 30 atg gaa agg gag ttc cct ggg ttt ttg gaa aat caa aag gac cctctg 201 Met Glu Arg Glu Phe Pro Gly Phe Leu Glu Asn Gln Lys Asp Pro Leu35 40 45 50 gct gtg gac aaa ata atg aaa gac ctg gac cag tgc cga gat ggaaaa 249 Ala Val Asp Lys Ile Met Lys Asp Leu Asp Gln Cys Arg Asp Gly Lys55 60 65 gtg ggc ttc cag agc ttt cta tca cta gtg gcg ggg ctc atc att gca297 Val Gly Phe Gln Ser Phe Leu Ser Leu Val Ala Gly Leu Ile Ile Ala 7075 80 tgc aat gac tat ttt gta gta cac atg aag cag aag aag taggccaact 346Cys Asn Asp Tyr Phe Val Val His Met Lys Gln Lys Lys 85 90 95 ggagccctggtacccacacc ttgatgcgtc ctctcccatg gggtcaactg aggaatctgc 406 cccactgcttcctgtgagca gatcaggacc cttaggaaat gtgcaaataa catccaactc 466 caattcgacaagcagagaaa gaaaagttaa tccaatgaca gaggagcttt cgagttttat 526 attgtttgcatccggttgcc ctcaataaag aaagtctttt tttttaagtt ccgaaaaaaa 586 aaaaaaaaaaaaaa 600 65 95 PRT Rattus sp. 65 Met Pro Ser Gln Met Glu His Ala Met GluThr Met Met Leu Thr Phe 1 5 10 15 His Arg Phe Ala Gly Glu Lys Asn TyrLeu Thr Lys Glu Asp Leu Arg 20 25 30 Val Leu Met Glu Arg Glu Phe Pro GlyPhe Leu Glu Asn Gln Lys Asp 35 40 45 Pro Leu Ala Val Asp Lys Ile Met LysAsp Leu Asp Gln Cys Arg Asp 50 55 60 Gly Lys Val Gly Phe Gln Ser Phe LeuSer Leu Val Ala Gly Leu Ile 65 70 75 80 Ile Ala Cys Asn Asp Tyr Phe ValVal His Met Lys Gln Lys Lys 85 90 95 66 639 DNA Rattus sp. CDS(1)..(636) 66 atg gcg tac gcc tat ctc ttc aag tac atc atc atc ggc gacaca ggt 48 Met Ala Tyr Ala Tyr Leu Phe Lys Tyr Ile Ile Ile Gly Asp ThrGly 1 5 10 15 gtt ggt aaa tcg tgc tta ttg cta cag ttt aca gac aag aggttt cag 96 Val Gly Lys Ser Cys Leu Leu Leu Gln Phe Thr Asp Lys Arg PheGln 20 25 30 ccg gtg cat gac ctc aca att ggt gta gag ttt ggt gct cga atgata 144 Pro Val His Asp Leu Thr Ile Gly Val Glu Phe Gly Ala Arg Met Ile35 40 45 acc att gat ggg aaa cag ata aaa ctc cag atc tgg gat aca gca ggg192 Thr Ile Asp Gly Lys Gln Ile Lys Leu Gln Ile Trp Asp Thr Ala Gly 5055 60 cag gag tcc ttt cgt tct atc aca agg tca tat tac aga ggt gca gcg240 Gln Glu Ser Phe Arg Ser Ile Thr Arg Ser Tyr Tyr Arg Gly Ala Ala 6570 75 80 ggg gct tta cta gtg tat gat att aca agg aga gac acg ttc aac cac288 Gly Ala Leu Leu Val Tyr Asp Ile Thr Arg Arg Asp Thr Phe Asn His 8590 95 ttg aca acc tgg tta gaa gac gcc cgt cag cat tcc aat tcc aac atg336 Leu Thr Thr Trp Leu Glu Asp Ala Arg Gln His Ser Asn Ser Asn Met 100105 110 gtc atc atg ctt att gga aat aaa agt gac tta gaa tct agg aga gaa384 Val Ile Met Leu Ile Gly Asn Lys Ser Asp Leu Glu Ser Arg Arg Glu 115120 125 gtg aaa aag gaa gaa ggt gaa gct ttt gca cga gag cat gga ctt atc432 Val Lys Lys Glu Glu Gly Glu Ala Phe Ala Arg Glu His Gly Leu Ile 130135 140 ttc atg gaa act tct gcc aag act gct tct aat gta gag gag gca ttt480 Phe Met Glu Thr Ser Ala Lys Thr Ala Ser Asn Val Glu Glu Ala Phe 145150 155 160 att aac aca gca aaa gaa att tat gaa aaa atc caa gaa ggg gtcttt 528 Ile Asn Thr Ala Lys Glu Ile Tyr Glu Lys Ile Gln Glu Gly Val Phe165 170 175 gac att aat aat gag gca aac ggc atc aaa att ggc cct cag catgct 576 Asp Ile Asn Asn Glu Ala Asn Gly Ile Lys Ile Gly Pro Gln His Ala180 185 190 gct acc aat gca tct cac gga ggc aac caa gga ggg cag cag gcaggg 624 Ala Thr Asn Ala Ser His Gly Gly Asn Gln Gly Gly Gln Gln Ala Gly195 200 205 gga ggc tgc tgc tga 639 Gly Gly Cys Cys 210 67 212 PRTRattus sp. 67 Met Ala Tyr Ala Tyr Leu Phe Lys Tyr Ile Ile Ile Gly AspThr Gly 1 5 10 15 Val Gly Lys Ser Cys Leu Leu Leu Gln Phe Thr Asp LysArg Phe Gln 20 25 30 Pro Val His Asp Leu Thr Ile Gly Val Glu Phe Gly AlaArg Met Ile 35 40 45 Thr Ile Asp Gly Lys Gln Ile Lys Leu Gln Ile Trp AspThr Ala Gly 50 55 60 Gln Glu Ser Phe Arg Ser Ile Thr Arg Ser Tyr Tyr ArgGly Ala Ala 65 70 75 80 Gly Ala Leu Leu Val Tyr Asp Ile Thr Arg Arg AspThr Phe Asn His 85 90 95 Leu Thr Thr Trp Leu Glu Asp Ala Arg Gln His SerAsn Ser Asn Met 100 105 110 Val Ile Met Leu Ile Gly Asn Lys Ser Asp LeuGlu Ser Arg Arg Glu 115 120 125 Val Lys Lys Glu Glu Gly Glu Ala Phe AlaArg Glu His Gly Leu Ile 130 135 140 Phe Met Glu Thr Ser Ala Lys Thr AlaSer Asn Val Glu Glu Ala Phe 145 150 155 160 Ile Asn Thr Ala Lys Glu IleTyr Glu Lys Ile Gln Glu Gly Val Phe 165 170 175 Asp Ile Asn Asn Glu AlaAsn Gly Ile Lys Ile Gly Pro Gln His Ala 180 185 190 Ala Thr Asn Ala SerHis Gly Gly Asn Gln Gly Gly Gln Gln Ala Gly 195 200 205 Gly Gly Cys Cys210 68 816 DNA Rattus sp. CDS (1)..(813) 68 atg gtg ctg ctc aag gaa tatcgg gtc atc ctg cct gtg tct gta gat 48 Met Val Leu Leu Lys Glu Tyr ArgVal Ile Leu Pro Val Ser Val Asp 1 5 10 15 gag tat caa gtg ggg cag ctgtac tct gtg gct gaa gcc agt aaa aat 96 Glu Tyr Gln Val Gly Gln Leu TyrSer Val Ala Glu Ala Ser Lys Asn 20 25 30 gaa act ggt ggt ggg gaa ggt gtggag gtc ctg gtg aac gag ccc tac 144 Glu Thr Gly Gly Gly Glu Gly Val GluVal Leu Val Asn Glu Pro Tyr 35 40 45 gag aag gat gat ggc gag aaa ggc cagtac aca cac aag atc tac cac 192 Glu Lys Asp Asp Gly Glu Lys Gly Gln TyrThr His Lys Ile Tyr His 50 55 60 tta cag agc aaa gtt ccc acg ttt gtt cgaatg ctg gcc cca gaa ggc 240 Leu Gln Ser Lys Val Pro Thr Phe Val Arg MetLeu Ala Pro Glu Gly 65 70 75 80 gcc ctg aat ata cat gag aaa gcc tgg aatgcc tac cct tac tgc aga 288 Ala Leu Asn Ile His Glu Lys Ala Trp Asn AlaTyr Pro Tyr Cys Arg 85 90 95 acc gtt att aca aat gag tac atg aag gaa gacttt ctc att aaa att 336 Thr Val Ile Thr Asn Glu Tyr Met Lys Glu Asp PheLeu Ile Lys Ile 100 105 110 gaa acc tgg cac aag cca gac ctt ggc acc caggag aat gtg cat aaa 384 Glu Thr Trp His Lys Pro Asp Leu Gly Thr Gln GluAsn Val His Lys 115 120 125 ctg gag cct gag gca tgg aaa cat gtg gaa gctata tat ata gac atc 432 Leu Glu Pro Glu Ala Trp Lys His Val Glu Ala IleTyr Ile Asp Ile 130 135 140 gct gat cga agc caa gta ctt agc aag gat tacaag gca gag gaa gac 480 Ala Asp Arg Ser Gln Val Leu Ser Lys Asp Tyr LysAla Glu Glu Asp 145 150 155 160 cca gca aaa ttt aaa tct atc aaa aca ggacga gga cca ttg ggc ccg 528 Pro Ala Lys Phe Lys Ser Ile Lys Thr Gly ArgGly Pro Leu Gly Pro 165 170 175 aat tgg aag caa gaa ctt gtc aat cag aaggac tgc cca tat atg tgt 576 Asn Trp Lys Gln Glu Leu Val Asn Gln Lys AspCys Pro Tyr Met Cys 180 185 190 gca tac aaa ctg gtt act gtc aag ttc aagtgg tgg ggc ttg cag aac 624 Ala Tyr Lys Leu Val Thr Val Lys Phe Lys TrpTrp Gly Leu Gln Asn 195 200 205 aaa gtg gaa aac ttt ata cat aag caa gagaag cgt ctg ttt aca aac 672 Lys Val Glu Asn Phe Ile His Lys Gln Glu LysArg Leu Phe Thr Asn 210 215 220 ttt cac agg cag ctg ttc tgt tgg ctt gataaa tgg gtt gat ctg act 720 Phe His Arg Gln Leu Phe Cys Trp Leu Asp LysTrp Val Asp Leu Thr 225 230 235 240 atg gat gac att cgg agg atg gaa gaagag acg aag aga cag ctg gat 768 Met Asp Asp Ile Arg Arg Met Glu Glu GluThr Lys Arg Gln Leu Asp 245 250 255 gag atg aga caa aag gac ccc gtg aaagga atg aca gca gat gac tag 816 Glu Met Arg Gln Lys Asp Pro Val Lys GlyMet Thr Ala Asp Asp 260 265 270 69 2263 DNA Simian sp. 69 cgctctcctcctcccctttc tctagcagta gccttcttaa tgtagtttaa tggctttaca 60 aagaaagccaggcagaggag cacttctcag tggctgtggt cggaccatga cctagctgac 120 catgaacttggaagggcttg aaatgatagc agttctgatc gtcattgtgc tttttgttaa 180 attattggaacagtttgggc tgattgaagc aggtttagaa gacagcgtgg aagatgaact 240 ggagatggccactgtcaggc atcggcctga ggcccttgag cttctggaag cccagagcaa 300 atttaccaagaaagagcttc agatccttta cagaggattt aagaacgaat gccccagtgg 360 tgttgttaatgaagaaacct tcaaagagat ttactcgcag ttctttccac agggagactc 420 tacaacatatgcacattttc tgttcaatgc gtttgatacg gaccacaatg gagctgtgag 480 tttcgaggatttcatcaaag gtctttccat tttgctccgg gggacagtac aagaaaaact 540 caattgggcatttaatctgt atgatataaa taaagatggc tacatcacta aagaggaaat 600 gcttgatataatgaaagcaa tatacgacat gatgggtaaa tgtacatatc ctgtcctcaa 660 agaagatgcacccagacaac acgtcgaaac attttttcag aaaatggaca aaaataaaga 720 tggggttgttaccatagatg agttcattga aagctgccaa aaagatgaaa acataatgcg 780 ctccatgcagctctttgaaa atgtgattta acttgtcaac tagatcctga atccaacaga 840 caaatgtgaactattctacc acccttaaag tcggagctac cacttttagc atagattgct 900 cagcttgacactgaagcata ttatgcaaac aagctttgtt ttaatataaa gcaatcccca 960 aaagatttgagtttctcagt tataaatttg catcctttcc ataatgccac tgagttcatg 1020 ggatgttctaactcatttca tactctgtga atattcaaaa gtaatagaat ctggcatata 1080 gttttattgattccttagcc atgggattat tgaggctttc acatatcagt gattttaaaa 1140 taccagtgttttttgctact catttgtatg tattcagtcc taggattttg aatggttttc 1200 taatatactgacatctgcat ttaatttcca gaaattaaat taattttcat gtctgaatgc 1260 tgtaattccatttatatact ttaagtaaac aaataagatt actacaatta aacacatagt 1320 tccagtttctatggccttca cttcccacct tctattagaa attaatttta tctggtattt 1380 ttaaacatttaaaaatttat catcagatat cagcatatgc ctaattatgc ctaatgaaac 1440 ttaataagcatttaattttc catcatacat tatagtcaag gcctatatac tatatataat 1500 tttggatttgtttaatctta caggctgttt tccattgtat catcaagtgg aagttcaaga 1560 cggcatcaaacaaaacaagg atgtttacag acatatgcaa agggtcagga tatctatcct 1620 ccagtatatgttaatgctta ataacaagta atcctaacag cattaaaggc caaatctgtc 1680 ctctttcccctgacttcctt acagcatgtt tatattacaa gccattcagg gacaaagaaa 1740 ccttgactaccccactgtct actaggaaca aacaaacagc aagcaaaatt cactttgaaa 1800 gcaccagtggttccattaca ttgacaacta ctaccaagat tcagtagaaa ataagtgctc 1860 aacaactaatccagattaca atatgattta gtgcatcata aaattccaac aattcagatt 1920 atttttaatcacctcagcca caactgtaaa gttgccacat tactaaagac acacacatcg 1980 tccctgttttgtagaaatat cacaaagacc aagaggctac agaaggagga aatttgcaac 2040 tgtctttgcaacaataaatc aggtatctat tctggtgtag agataggatg ttgaaagctg 2100 ccctgctatcaccagtgtag aaattaagag tagtacaata catgtacact gaaatttgcc 2160 atcgcgtgtttgtgtaaact caatgtgcac attttgtatt tcaaaaagaa aaaataaaag 2220 caaaataaaatgtttataac tctaaaaaaa aaaaaaaaaa aaa 2263 70 229 PRT Simian sp. 70 MetAsn Leu Glu Gly Leu Glu Met Ile Ala Val Leu Ile Val Ile Val 1 5 10 15Leu Phe Val Lys Leu Leu Glu Gln Phe Gly Leu Ile Glu Ala Gly Leu 20 25 30Glu Asp Ser Val Glu Asp Glu Leu Glu Met Ala Thr Val Arg His Arg 35 40 45Pro Glu Ala Leu Glu Leu Leu Glu Ala Gln Ser Lys Phe Thr Lys Lys 50 55 60Glu Leu Gln Ile Leu Tyr Arg Gly Phe Lys Asn Glu Cys Pro Ser Gly 65 70 7580 Val Val Asn Glu Glu Thr Phe Lys Glu Ile Tyr Ser Gln Phe Phe Pro 85 9095 Gln Gly Asp Ser Thr Thr Tyr Ala His Phe Leu Phe Asn Ala Phe Asp 100105 110 Thr Asp His Asn Gly Ala Val Ser Phe Glu Asp Phe Ile Lys Gly Leu115 120 125 Ser Ile Leu Leu Arg Gly Thr Val Gln Glu Lys Leu Asn Trp AlaPhe 130 135 140 Asn Leu Tyr Asp Ile Asn Lys Asp Gly Tyr Ile Thr Lys GluGlu Met 145 150 155 160 Leu Asp Ile Met Lys Ala Ile Tyr Asp Met Met GlyLys Cys Thr Tyr 165 170 175 Pro Val Leu Lys Glu Asp Ala Pro Arg Gln HisVal Glu Thr Phe Phe 180 185 190 Gln Lys Met Asp Lys Asn Lys Asp Gly ValVal Thr Ile Asp Glu Phe 195 200 205 Ile Glu Ser Cys Gln Lys Asp Glu AsnIle Met Arg Ser Met Gln Leu 210 215 220 Phe Glu Asn Val Ile 225 71 2259DNA Simian sp. 71 gtcgacagac gcccctggcc ggtggactcc tgagtcttac tcctgcaccctgcgtcccca 60 gacatgaatg tgaggagagt ggaaagcatt tcggctcagc tggaggaggccagctccaca 120 ggcggtttcc tgtatgctca gaacagcacc aagcgcagca ttaaagagcggctcatgaag 180 ctcttgccct gctcagctgc caaaacatcg tctcctgcta ttcaaaacagcgtggaagat 240 gaactggaga tggccactgt caggcatcgg cctgaggccc ttgagcttctggaagcccag 300 agcaaattta ccaagaaaga gcttcagatc ctttacagag gatttaagaacgaatgcccc 360 agtggtgttg ttaatgaaga aaccttcaaa gagatttact cgcagttctttccacaggga 420 gactctacaa catatgcaca ttttctgttc aatgcgtttg atacggaccacaatggagct 480 gtgagtttcg aggatttcat caaaggtctt tccattttgc tccgggggacagtacaagaa 540 aaactcaatt gggcatttaa tctgtatgat ataaataaag atggctacatcactaaagag 600 gaaatgcttg atataatgaa agcaatatac gacatgatgg gtaaatgtacatatcctgtc 660 ctcaaagaag atgcacccag acaacacgtc gaaacatttt ttcagaaaatggacaaaaat 720 aaagatgggg ttgttaccat agatgagttc attgaaagct gccaaaaagatgaaaacata 780 atgcgctcca tgcagctctt tgaaaatgtg atttaacttg tcaactagatcctgaatcca 840 acagacaaat gtgaactatt ctaccaccct taaagtcgga gctaccacttttagcataga 900 ttgctcagct tgacactgaa gcatattatg caaacaagct ttgttttaatataaagcaat 960 ccccaaaaga tttgagtttc tcagttataa atttgcatcc tttccataatgccactgagt 1020 tcatgggatg ttctgactca tttcatactc tgtgaatatt caaaagtaatagaatctggc 1080 atatagtttt attgattcct tagccatggg attattgagg ctttcacatatcagtgattt 1140 taaaatacca gtgttttttg ctactcattt gtatgtattc agtcctaggattttgaatgg 1200 ttttctaata tactgacatc tgcatttaat ttccagaaat taaattaattttcatgtctg 1260 aatgctgtaa ttccatttat atactttaag taaacaaata agattactacaattaaacac 1320 atagttccag tttctatggc cttcacttcc caccttctat tagaaattaattttatctgg 1380 tatttttaaa catttaaaaa tttatcatca gatatcagca tatgcctaattatgcctaat 1440 gaaacttaat aagcatttaa ttttccatca tacattatag tcaaggcctatatactatat 1500 ataattttgg atttgtttaa tcttacaggc tgttttccat tgtatcatcaagtggaagtt 1560 caagacggca tcaaacaaaa caaggatgtt tacagacata tgcaaagggtcaggatatct 1620 atcctccagt atatgttaat gcttaataac aagtaatcct aacagcattaaaggccaaat 1680 ctgtcctctt tcccctgact tccttacagc atgtttatat tacaagccattcagggacaa 1740 agaaaccttg actaccccac tgtctactag gaacaaacaa acagcaagcaaaattcactt 1800 tgaaagcacc agtggttcca ttacattgac aactactacc aagattcagtagaaaataag 1860 tgctcaacaa ctaatccaga ttacaatatg atttagtgca tcataaaattccaacaattc 1920 agattatttt taatcacctc agccacaact gtaaagttgc cacattactaaagacacaca 1980 catcgtccct gttttgtaga aatatcacaa agaccaagag gctacagaaggaggaaattt 2040 gcaactgtct ttgcaacaat aaatcaggta tctattctgg tgtagagataggatgttgaa 2100 agctgccctg ctatcaccag tgtagaaatt aagagtagta caatacatgtacactgaaat 2160 ttgccatcgc gtgtttgtgt aaactcaatg tgcacatttt gtatttcaaaaagaaaaaat 2220 aaaagcaaaa taaaatgtta aaaaaaaaaa aaaaaaaaa 2259 72 250PRT Simian sp. 72 Met Asn Val Arg Arg Val Glu Ser Ile Ser Ala Gln LeuGlu Glu Ala 1 5 10 15 Ser Ser Thr Gly Gly Phe Leu Tyr Ala Gln Asn SerThr Lys Arg Ser 20 25 30 Ile Lys Glu Arg Leu Met Lys Leu Leu Pro Cys SerAla Ala Lys Thr 35 40 45 Ser Ser Pro Ala Ile Gln Asn Ser Val Glu Asp GluLeu Glu Met Ala 50 55 60 Thr Val Arg His Arg Pro Glu Ala Leu Glu Leu LeuGlu Ala Gln Ser 65 70 75 80 Lys Phe Thr Lys Lys Glu Leu Gln Ile Leu TyrArg Gly Phe Lys Asn 85 90 95 Glu Cys Pro Ser Gly Val Val Asn Glu Glu ThrPhe Lys Glu Ile Tyr 100 105 110 Ser Gln Phe Phe Pro Gln Gly Asp Ser ThrThr Tyr Ala His Phe Leu 115 120 125 Phe Asn Ala Phe Asp Thr Asp His AsnGly Ala Val Ser Phe Glu Asp 130 135 140 Phe Ile Lys Gly Leu Ser Ile LeuLeu Arg Gly Thr Val Gln Glu Lys 145 150 155 160 Leu Asn Trp Ala Phe AsnLeu Tyr Asp Ile Asn Lys Asp Gly Tyr Ile 165 170 175 Thr Lys Glu Glu MetLeu Asp Ile Met Lys Ala Ile Tyr Asp Met Met 180 185 190 Gly Lys Cys ThrTyr Pro Val Leu Lys Glu Asp Ala Pro Arg Gln His 195 200 205 Val Glu ThrPhe Phe Gln Lys Met Asp Lys Asn Lys Asp Gly Val Val 210 215 220 Thr IleAsp Glu Phe Ile Glu Ser Cys Gln Lys Asp Glu Asn Ile Met 225 230 235 240Arg Ser Met Gln Leu Phe Glu Asn Val Ile 245 250 73 10 PRT Simian sp. 73Ser Asn Ala Lys Ala Val Glu Thr Asp Val 1 5 10

What is claimed is:
 1. An isolated nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:19, SEQ IDNO:23, SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:52, or a complementthereof.
 2. An isolated nucleic acid molecule consisting of thenucleotide sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:19, SEQ IDNO:23, SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:52, or complementthereof.
 3. An isolated nucleic acid molecule comprising the nucleotidesequence of the DNA insert of the plasmid deposited with ATCC asAccession Number 98994, 98993, 98991, 98951, or PTA-316, or a complementthereof.
 4. An isolated nucleic acid molecule consisting of thenucleotide sequence of the DNA insert of the plasmid deposited with ATCCas Accession Number 98994, 98993, 98991, 98951, or PTA-316, or acomplement thereof.
 5. An isolated nucleic acid molecule which ecodes apolypepride comprising the amino acid sequence of SEQ ID NO:2, SEQ IDNO:14, SEQ ID NO:20, SEQ ID NO:24, or SEQ ID NO:53.
 6. An isolatednucleic acid molecule which encodes a polypepride consisting of aminoacid sequence of SEQ ID NO:2, SEQ ID NO:14, SEQ ID NO:20, SEQ ID NO:24,or SEQ ID NO:53.
 7. An isolated nucleic acid molecule which encodes apolypeptide comprising amino acid residues 32-216 of the amino acidsequence of SEQ ID NO:2.
 8. An isolated nucleic acid molecule whichencodes a polypeptide comprising amino acid residues 68-270 of the aminoacid sequence of SEQ ID NO:14.
 9. The nucleic acid molecule of any oneof claims 1, 2, 3, 4, 5, 6, 7 or 8, further comprising vector nucleicacid sequences.
 10. The nucleic acid molecule of any one of claims 1, 2,3, 4, 5, 6, 7 or 8, further comprising nucleic acid sequences encoding aheterologous polypeptide.
 11. An isolated host cell which comprises thenucleic acid molecule of any one of claims 1, 2, 3, 4, 5, 6, 7 or
 8. 12.The host cell of claim 11 which is a mammalian host cell.
 13. A methodfor producing a polypeptide comprising culturing the host cell of claim11 under conditions in which the nucleic acid molecule is expressed.